Survey for potentially necrotizing spider venoms, with special emphasis on Cheiracanthium mildei

Comparative Biochemistry and Physiology, Part C 141 (2005) 32 – 39 www.elsevier.com/locate/cbpc

Survey for potentially necrotizing spider venoms, with special emphasis on Cheiracanthium mildei Matthew J. Foradoria,*, Samuel C. Smithb, Elizabeth Smithb, Roger E. Wellsb b

a Department of Zoology, University of New Hampshire, Durham, NH 03824, USA Department of Animal, Nutritional, and Medical Laboratory Sciences, University of New Hampshire, Durham, NH 03824, USA

Received 31 December 2004; received in revised form 2 May 2005; accepted 2 May 2005 Available online 23 June 2005

Abstract It has proven difficult to identify those spiders which cause necrotic lesions. In an effort to design a simple, inexpensive screening method for identifying spiders with necrotizing venoms, we have examined the venom gland homogenates of a variety of spider species for their ability to cause red blood cell lysis. Those venoms which were positive were further examined for the presence of sphingomyelinase D, and their ability to evoke necrotic lesions in the skin of rabbits. Sphingomyelinase D is known to be the causative agent of necrosis and red blood cell lysis in the venom of the brown recluse spider (Loxosceles reclusa), and our assumption was that this would be the same agent in other spider venoms as well. This did not prove to be the case. Of 45 species examined, only the venom of L. reclusa and Cheiracanthium mildei lysed sheep red blood cells. Unlike L. reclusa venom, however, C. mildei venom did not possess sphingomyelinase D nor did it cause necrotic lesions in the skin of rabbits. We present evidence suggesting that a phospholipase A2 is the hemolytic agent in C. mildei venom. D 2005 Elsevier Inc. All rights reserved. Keywords: Cheiracanthium; Hemolysis; Loxosceles; Necrosis; Necrotic arachnidism; Phosphatidylcholine; Phospholipase A2; Venom gland homogenate

1. Introduction It has proven difficult to identify which spider species produce necrotic lesions. Only rarely does a person seeking medical attention for a spider bite have the specimen in their possession. In the absence of the culprit, it is nearly impossible to distinguish spider bites from those produced by other arthropods (Russell and Gertsch, 1983) or even some disease states (Vetter, 2000). It is also difficult for arachnologists to link particular spider species directly with clinically significant bites. First, it is necessary to inspect the community where suspected spider bites occurred to confirm that the spider exists in sufficient numbers as to suggest a probable cause. In addition, it is necessary to demonstrate that the injection of the venom will produce the appropriate lesions (Speilman and Levi, 1970; Vest,

* Corresponding author. Tel.: +1 603 862 1791; fax: +1 603 862 3784. E-mail address: [email protected] (M.J. Foradori). 1532-0456/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2005.05.001

1987a,b). And whereas reviews listing spider species that potentially cause lesions have been assembled (Russell and Gertsch, 1983; White et al., 1995; Vetter and Visscher, 1998; Sams et al., 2001), the lists continue to be modified. The continuing lack of consensus has led White (1999) to remark that ‘‘a concerted effort to determine which species can cause necrotic arachnidism should become a prime focus for research in this field’’. It is now well established that envenomation by the genus Loxosceles (brown spiders), known as loxoscelism, is capable of causing severe necrotic lesions. The first link between a brown spider bite and tissue death was attributed to the South American brown spider, Loxosceles laeta, by Macchiavello (1937); a link with the brown recluse, L. reclusa was first established in the United States by Atkins et al. (1958). In a systematic search for the necrotizing agent in the venom of L. reclusa, Forrester et al. (1978) found sphingomyelinase D to be the hemolytic constituent. The isolated enzyme was shown to cause necrotic lesions in rabbits (Kurpiewski et al., 1981), and antibodies to the

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purified enzyme were later shown to prevent necrosis (Gomez et al., 1999). These observations have since been extended to other South American Loxosceles species (Barbaro et al., 1994; Tambourgi et al., 1998; Guilherme et al., 2001; for a current and comprehensive review of Loxoscelism, please refer to Futrell, 1992, and da Silva et al., 2004). In an effort to develop a simple, inexpensive screening method to identify those spiders which are capable of causing necrotic lesions, we have examined the venom of a variety of spiders for the ability to cause sheep red blood cell (RBC) hemolysis. Those that were positive were further screened for sphingomyelinase D and dermonecrotic activity. Our survey was based upon the assumption that those spider venoms that cause necrosis would also possess sphingomyelinase D and, therefore, cause sheep RBC lysis. This did not prove to be the case; of 45 spider species surveyed, only the venom of L. reclusa and C. mildei lysed sheep red blood cells. Unlike L. reclusa venom, however, C. mildei venom did not possess sphingomyelinase D, nor did it cause necrotic lesions in the skin of rabbits. We present evidence suggesting that a calcium-dependent phospholipase A2 is the hemolytic agent in C. mildei venom.

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three days post-capture to ensure venom glands were not depleted. Spiders were narcotized by carbon dioxide, and venom glands were removed by micro-dissection. The glands were either used immediately or frozen for future use. 2.3. Hemolysis assay Hemolytic activity was determined using the spectrophotometric method of Forrester et al. (1978). One venom gland from each spider used was homogenized in 50 AL of a 20 mM Tris buffered saline with 8 mM CaCl2, pH 7.4 (TBSC; after Babcock et al., 1981). The entire venom gland homogenate (VGH) was added to 1.0 mL suspension of a 2% sheep red blood cells in a TBSC suspension and incubated at 37 -C for 1.5 h with gentle agitation. The suspension was then diluted with 3 mL TBSC and centrifuged at approximately 760 g for 5 min. The optical densities of the supernatants were measured at 540 nm with a Spectronic 601 spectrophotometer (Milton-Roy). Within each assay, a 1 mL aliquot of the sheep RBC suspension was incubated as stated above, then lysed with 3 mL distilled water, and used as the standard for 100% hemolysis. Percent hemolysis was determined against total hemolysis.

2. Materials and methods 2.4. Sphingomyelinase assay 2.1. Reagents Defibrinated sheep blood was obtained from Northeast Laboratory (Waterville, ME). N-Trinitrophenylaminolauric acid (TNPAL)-sphingomyelin (T 1014), sphingomyelinase D from Staphylococcus aureus (S 8633), snake venom from Crotalus atrox (V 7000), and ethylene glycol-bis (2aminoethylether)-N,N,NV,NV-tetraacetic acid (EGTA, E 3889) were purchased from the Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Silica Gel 60 TLC plates (5721-7) were purchased from EM Sciences (Cincinnati, OH, USA). Purified phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SM) were acquired as a phosphoglycerides kit (#1053) from Matreya, Inc. (State College, PA, USA). BODIPY-labeled phosphatidylcholines 2-(4,4-difluoro-5,7-dimethyl-4-bora3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-snglycero-3-phosphocholine (D-3803) and 2-decanoyl-1-(O(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)undecyl)-sn-glycero-3-phosphocholine) (D-3771) were purchased from Invitrogen (Carlsbad, CA, USA). 2.2. Preparation of venom gland homogenates All spiders were collected between June 2001 and November 2002 in the vicinity of Durham, New Hampshire, unless otherwise noted. The spiders were fasted for at least

Sphingomyelinase D activity was determined using the spectrophotometric method of Gatt et al. (1978) as modified by Young and Pincus (2001) using (TNPAL)sphingomyelin as substrates. Each venom gland was homogenized in 30 AL 20 mM HEPES with 8 mM CaCl2 pH 7.1 then added to 170 AL solution of 0.3 mM TNPALsphingomyelin containing 1.5 mM Triton X-100 in the above buffer. All samples were incubated at 37 -C for 2 h. The positive control substituted 1.0 unit of sphingomyelinase D from S. aureus in place of spider VGH. Reactions were stopped by the addition of 750 AL of isopropanol : heptane : 5 M sulfuric acid (40 : 10 : 1 v/v). The mixtures were extracted with 450 AL heptane, followed by 400 AL distilled H2O. All samples were vortexed then centrifuged for 10 min. The upper heptane phase was removed and its absorbance was read at 330 nm. 2.5. Phospholipase assays Defibrinated sheep RBC ghosts were prepared according to Dodge et al. (1963). Individual venom glands from spiders were homogenized in 50 AL of 20 mM HEPES buffer pH 7.1 with 8 mM CaCl2. The VGH was then added to 500 AL of a 17% RBC ghost suspension in the same buffer, and incubated for 3 h at 37 -C. Eight Ag protein of snake venom from Crotalus atrox were incubated with the same amount of the RBC ghost suspension as a positive control.

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Individual phosphoglycerides were used to determine the substrate specificity of the hemolytic factor found in C. mildei VGH. One hundred fifty Ag of purified phosphatidylethanolamine, phosphatidylserine, or phosphatidylcholine in 500 AL of 20 mM HEPES buffer pH 7.1 with 8 mM CaCl2, were incubated with one homogenized venom gland in 50 AL of the above buffer for 2 h at 37 -C. Membrane phospholipids and cleavage products were extracted with chloroform : methanol (2 : 1 v/v) and separated on Silica Gel 60 TLC using Solvent B (chloroform : methanol : 2-propanol : 0.25% potassium chloride : triethylamine (30 : 9 : 25 : 6 : 18 v/v)) of Touchstone et al. (1980). Results were visualized by staining with 3% cupric acetate, 8% phosphoric acid solution then heating at 180 -C for 10 min. Phospholipase A activity was determined by the method of Farber et al. (1999). BODIPY\-FL labeled phosphatidylcholine was used to determine whether the enzyme cleaved the fatty acid from the sn-1 (D-3771) or sn-2 (D3803) position. Cleavage products were separated by TLC methods using solvent B of Touchstone et al. (1980) and visualized with a 488 nm laser on a Bio-Rad Molecular Imager FX Pro. 2.6. The effect of C. mildei venom gland homogenate on the skin of rabbits To observe if venom from C. mildei is capable of causing necrotic lesions VGH was injected into the skin of rabbits. The experiments were carried out in compliance with the Public Health Service Policy, the Guide for the Care and Use of Laboratory Animals (1996), and University Policy. Three adult female NZW rabbits (Oryctolagus cuniculus) were injected in a shaved area on the back with the following solutions: site I: 200 AL PBS, pH 7.4; site II: 15 Ag of C. mildei VGH protein in 200 AL PBS. All preparations were filtered through a 0.2 Am filter prior to injection. The injections were administered intradermally with a 26 gauge needle approximately 10 –12 cm apart. The rabbits were observed hourly for the first 6 h and then every 12 h for the next 138 h. The injection sites were photographed with a digital camera and scored according to the intracutaneous test described in the USP 24-NF19 (2000). The same rabbits were administered a 5-fold increase in concentration of C. mildei VGH protein seven days later. Site I again served as the control, being 200 AL PBS. Site II was moved to the opposite side of the freshly shaved rabbit. The injectate contained 79 Ag of C. mildei VGH protein (approximately 4.4 venom glands) in 200 AL PBS. The rabbits were observed hourly for the first 6 h and then every 12 h for 48 h. Rabbits were euthanized and skin samples were treated as described in the methods of Foradori et al. (2001). For comparative purposes we are including photographs of our results from an earlier study showing the effect of L. reclusa VGH on the skin of rabbits (Foradori et al., 2001).

Table 1 Survey of the venom of selected spider species for their ability to cause hemolysis Family

Species

Loxosceles reclusa (3)t Chiracanthium mildei female (9) male (8) Gnaphosidae Herpyllus ecclesiastices (2) Micaria longipes (2) Unidentified Gnaphosidae (6) Dtsderidae Dysdera crocota (1)n Salticidae Phidippus audax (8) Phidippus clarus (2) Platycryptus undatus (4) Salticus scenicus (1) Unidentified Salticidae (5) Tetragnathidae Tetragnatha elongata (6) Nephila clavipes (4)f Araneidae Argiope aurantia (4) Argiope trifasciata (6) Larinoides cornutus (10)p Neoscona arabesca (5) Araneus cavaticus (4) Araneus diadematus (3) Araneus marmoreus (4) Araneus trifolium (8) Gastercantha cancriformis (8)f Micrathena sagittata (4)g Micrathena gracilis (3)g Metazyga sp. (5) Verrucosa sp. (3)f Unidentified Araneidae (9) Agelenidae Agelenopsis aperta (4)v Unidentified Agelenopsis (10) Filistatidae Kukulcania hibernalis (6)f Scytodidae Scytodes thoracica (2) Pholcidae Pholcus phalangoides (4) Mimetidae Mimetus sp. (5) Theridiidae Steatoda borealis (7) Theridion frondeum (3) Achaearanea tepidariorum (8) Unidentified Theridiidae (3) Latrodectus geometricus (4)f Latrodectus mactans (3) Pisauridae Pisaurina mira (7) Dolomedes tenebrosus (3) Dolomedes triton (3) Dolomedes vittatus (3) Unidentified Pisauridae Thomisidae Misumeops vatia (3) Misumeops asperatus (1) Tibellus sp (5) Unidentified Thomisidae Oxyopidae Oxyopes salticus salticus (3)k Peucetia viridans (3)f Linyphiidae Frontinella sp (5) Lycosidae Gladicosa gulosa (1) Unidentified Lycosidae (3) Loxoscelidae Miturgidae

Sheep RBC Hemolysis ++ ++ + -

One venom gland was homogenized from each spider and assayed for its ability to cause hemolysis in 1.0 mL of 2% sheep RBC suspension. Number of spiders tested is indicated in parentheses. All spiders tested were female unless noted otherwise. All spiders were collected in New Hampshire unless otherwise noted (f=Florida; g=Georgia; k=Kansas; n=North Carolina; p=Pennsylvania; t=Texas; v=Virginia).

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comparison was repeated three times and the data were evaluated using a t-test.

3. Results 3.1. Hemolysis assay C. mildei was the only VGH in this survey found to cause sheep RBC hemolysis (Table 1), other than L. reclusa, which was used as the positive control. C. mildei females caused an average of 65.4% hemolysis; males 38.9% hemolysis. L. reclusa females caused 37.5% hemolysis. The VGH of all other spiders surveyed showed little (< 0.5%) or no ability to cause hemolysis. 3.2. Sphingomyelinase assay

Fig. 1. The effects of spider venom gland homogenates (VGH) on RBC ghosts. Extracts of RBC ghosts treated with VGH were chromatographed in the one-dimensional system of Touchstone et al. (1980). The main components were phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI), and sphingomyelin (SM). RBC ghosts treated with Cheiracanthium mildei VGH had a decreased density of PE, and PS, and the appearance of free fatty acids (FA), as well as lysoforms (LP) of the previously mentioned phospholipids. Note the complete disappearance of SM with Loxosceles reclusa VGH. (Spiders from left to right are: C. mildei, Phidippus audax, Latrodectus geometricus, and L. reclusa).

2.7. The calcium dependency of the hemolytic factor in C. mildei venom To observe whether calcium was essential to the hemolytic factor, individual venom glands were homogenized in 1.0 mL 2% sheep RBC in 20 mM Tris buffered saline, pH 7.4 (300 mOsm) with or without 8 mM CaCl2. Hemolysis was evaluated as described above (part 2.3). The

We did not observe any sphingomyelinase D activity in female C. mildei VGH using the modified method of Gatt et al. (1978), whereas L. reclusa VGH homogenate was rich in this enzyme. And while L. reclusa VGH cleaved the sphingomyelin of RBC ghosts completely, the VGH of C. mildei had little or no effect on this substrate (Fig. 1). 3.3. Phospholipase assay The analysis of cleavage products from RBC ghosts exposed to venom gland homogenates of C. mildei revealed the release of free fatty acids signaling the presence of a phospholipase (Fig. 1). Phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) all released a free fatty acid and lysophospholipid when exposed to VGH of C. mildei (Fig. 2) indicating the presence of a Phospholipase A. Phosphatidylcholine fluorescently labeled on the sn-1 position released fluorescent labeled lysophosphatidylcholine;

Fig. 2. The effect of Cheiracanthium mildei venom gland homogenate (VGH) on individual purified phospholipids: Phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PC). Snake venom (Crotalus atrox) was used as a positive control.

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Fig. 3. The effect of Cheiracanthium mildei venom gland homogenate (VGH) on phosphatidylcholine ( fPC) fluorescently labeled on the sn-1 (D-3771) or sn-2 (D-3803) position. The release of fluorescent fatty acid ( fFA) from D-3803 and fluorescent lysophosphatidylcholine ( fLPC) from D-3771 indicate the presence of phospholipase A2 in the VGH.

phosphatidylcholine fluorescently labeled on the sn-2 (D3803) position released fluorescently labeled free fatty acid upon exposure to C. mildei venom. These observations specifically indicate the presence of a phospholipase A2 (Fig. 3). 3.4. The effect of C. mildei venom gland homogenate on the skin of rabbits A six mm diameter red flare appeared consistently within an hour of the C. mildei VGH injection. This flare began to regress within 24 h (Fig. 4B). The PBS control produced no observable changes in the skin and subcutis.

The microscopic appearance of the injection sites was consistent in all three rabbits. Histological sections of skin and subcutis of NZW rabbits injected intradermally with 79 Ag protein C. mildei VGH showed only a slight reaction in comparison with the lesions which we observed earlier in rabbits injected of 10 Ag protein L. reclusa VGH (Fig. 4C; from Foradori et al., 2001). Microscopic examination of the 5-fold increases of C. mildei VGH protein injection site revealed a well demarcated and focal reaction with edema and mixed inflammatory cell infiltrates, and mild necrosis in the panniculus, around follicles, and in the surrounding subcutis (Fig. 5A1, A2). Compared to the severe necrosis induced by 10 Ag protein

Fig. 4. The effect of Cheiracanthium mildei venom gland homogenate (VGH) on the skin of New Zealand white rabbits. A. Control injection with PBS. B. C. mildei VGH (79 Ag protein), and C. Loxosceles reclusa VGH (10 Ag protein). All injections were made intradermally and all photographs taken at 48 h postinjection. Unlabelled dark spots are permanent marker used to locate the injection sites. The photograph of L. reclusa was taken during an earlier study (Foradori et al., 2001) and is presented for comparison with those of C. mildei.

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Fig. 5. Histology of the skin of New Zealand white rabbit 48 hours after intradermal injection of 79 Ag protein Cheiracanthium mildei venom gland homogenate. A1. View of the dermis and epidermis reveals edema (e) and hair follicles (asterisks) showing heavy heterophil infiltration (2.5 objective); A2. Closer inspection shows mixed inflammatory cell infiltrates and mild necrosis in the panniculus (p) and subcutis (20 objective). B1 and B2 (2.5 and 20 objectives, respectively) are photomicrographs made 48 h after intradermal injection of 10 Ag protein L. reclusa VGH (from the study of Foradori et al., 2001). Notice the severity of tissue disruption resulting from 10 Ag compared to that produced by 79 Ag protein C. mildei venom gland homogenate.

VGH injections from L. reclusa (Fig. 5B1, B2; from Foradori et al., 2001), only minimal necrosis was observed when 8 times the protein concentration of C. mildei VGH was injected. 3.5. The calcium dependency of the hemolytic factor in C. mildei venom gland homogenate The absence of calcium significantly reduced the hemolytic capacity of the enzyme by almost 3-fold ( p  0.001) as seen in Fig. 6.

Fig. 6. Calcium dependency of Cheiracanthium mildei venom gland homogenate hemolytic factor. Homogenates of individual venom glands were incubated with 1.0 mL 2% sheep RBC in 20 mM Tris buffered saline, pH 7.4 (300 mOsm) with or without 8 mM CaCl2. (n = 3; error bars = SEM)

4. Discussion 4.1. Venom survey The VGH of a variety of spiders has been surveyed for their ability to cause hemolysis of sheep erythrocytes as a potentially simple and inexpensive way to screen for necrotizing spider venoms. This survey was based upon the assumption that such venoms would contain sphingomyelinase D, the necrotizing component in the venom of the brown recluse (Kurpiewski et al., 1981), which is also known to cause RBC lysis (Forrester et al., 1978). Of the 45 spider species examined, representing 20 spider families, only the VGH of C. mildei and L. reclusa were observed to cause hemolysis (Table 1). Our results for C. mildei accord with those of Croucamp and Veale (1999), who observed hemolytic activity in the venom of C. furculatum. However, in contrast to L. reclusa, C. mildei VGH did not possess sphingomyelinase D. Hemolytic factors, other than sphingomyelinase D are known to exist in the venom of some spiders. Amphipathic peptides that are both antimicrobial and hemolytic have been found in the venom of lycosids (Yan and Adams, 1998), oxyopids (Corzo et al., 2002), and ctenids (KuhnNentwig et al., 2002a,b), and appear to be universal components in spider venoms. To reduce their possible interference with our hemolysis assay we used a single venom gland in 1.0 mL of 2% sheep RBC in the assay. Sheep RBC are known to be much less susceptible to amphipathic peptides than those of the guinea pig and pig

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and a higher concentration of amphipathic peptides are required for lysis (Corzo et al., 2002). This precaution appears to have worked, as we observed lysis with the venom of only two spider species of the 45 surveyed (Table 1) and both can be explained by the presence of hydrolytic enzymes. However, a possible contribution of amphipathic peptides to the RBC lysis observed with C. mildei nor L. reclusa VGH has not yet been ruled out. Cohen and Quistad (1998) observed that the venom of P. audax contained a potent cytolytic factor that disrupted cells in culture. However, we observed neither lysis of erythrocytes nor cleavage products when the VGH of P. audax was incubated with RBC ghosts (Fig. 1). 4.2. Hemolytic factor in the venom of C. mildei To establish the factor present in the VGH of C. mildei responsible for RBC lysis, filtered VGHs were incubated with sheep RBC ghosts. Thin layer chromatography of filtered VGH incubated with RBC ghost extracts revealed a decreased density of PE, PS, and PI as well as an appearance of fatty acids and lysoforms of the lipids, indicating the presence of a phospholipase (Fig. 1). Incubation of C. mildei VGH with commercially available PE, PS, and PC confirmed the cleavage of these compounds (Fig. 2). To identify the site of cleavage of the phospholipids, VGH was incubated with PC fluorescently labeled on the sn-1 (D-3771) or sn-2 position (D3803). Whereas fluorescently labeled fatty acid was released from D-3803, fluorescently labeled lysophospholipid was released from D-3771 (Fig. 3) suggesting the presence of a phospholipase A2 in the venom gland homogenates of C. mildei. The activity of the hemolytic agent was reduced by the absence of exogenous Ca2+. This observation is consistent with the presence of a calciumdependent phospholipase A2 (secretory PLA2 (sPLA2); Akiba and Sato, 2004). PLA2 has been observed in the venom of the spider Eresus cinnaberinus (=niger) and has been shown to produce an irreversible blockage of frog neuromuscular junctions, as well as anticoagulant activity (Usmanov and Nuritova, 1994). Secretory PLA2 is a hemolytic agent also present in the venom of most snakes (Tu, 1977), and like the snake venom enzyme, C. mildei venom gland homogenate PLA2 is calcium-dependent (Fig. 6). 4.3. Pathology of C. mildei venom The venom of C. mildei has been observed to cause necrotic lesions in the skin of guinea pigs (Speilman and Levi, 1970) and hamsters (Hughes, 1981), and Cheiracanthium lawrencei venom has been shown to cause necrosis in the skin of rabbits (Newlands et al., 1980). We, however, observed only a mild erythema in the skin of rabbits following the injection of the equivalent of 4 venom glands (Fig. 4). Speilman and Levi (1970), Hughes (1981),

and Newlands et al. (1980) allowed spiders to bite their prey, whereas we injected the rabbits intradermally with VGH. The difference in the technique may account for the differences in the results obtained. And whereas Newlands et al. (1980) observed ulcerating lesions in humans resulting from the envenomation of C. lawrencei, most observations suggest that the effects of Cheiracanthium spider bites in humans are mild. Speilman and Levi (1970) observed only a mild reddening of the skin of a human volunteer resulting from C. mildei spider bite. Minton (1972) and Krinsky (1987) also observed only redness, swelling, and itching in humans resulting from the bite of this species. Similar mild reactions have been observed for the venom of C. inclusum (Furman and Reeves, 1957; Gorham and Rheney, 1968; Leech and Brown, 1994), C. punctorium (Maretic, 1982), and C. japonicum (Owri, 1978). From this study, it would appear that spider venoms containing sphingomyelinase D are rare, at least among North American species. Of all the spiders surveyed, only L. reclusa and C. mildei venoms were observed to cleave sheep red blood cells. And whereas the active agent in L. reclusa venom is sphingomyelinase D, it appears to be a phospholipase A2 in the venom of C. mildei. In contrast with the severe necrosis caused by 10 Ag VGH protein of L. reclusa (Foradori et al., 2001), we observed only a slight necrosis using the equivalent of 4.4 venom glands (79 Ag protein) from C. mildei.

Acknowledgments We want to thank Dr. Edward K. Tillinghast for advice and support throughout this study. We thank also Ms. Lauren Kiel for helping during the initial stages of the study, Ms. Becky Sink for providing brown recluse, and Dr. Bruce Cutler for Oxyopes salticus salticus. The research was supported by grant R15DK/OD51291 from the National Institutes of Health.

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