Clinical Toxicology (2007) 45, 678–687 Copyright © Informa Healthcare USA, Inc.ISSN: 1556-3650 print / 1556-9519 onlineDOI: 10.1080/15563650701397001

LCLTARTICLE

Toxicity of two North American Loxosceles (brown recluse spiders) venoms and their neutralization by antivenoms

Loxosceles venoms and antivenomsADOLFO RAFAEL DE ROODT, PH.D.1, JUDITH ESTEVEZ-RAMÍREZ, M.SC.2, SILVANA LITWIN, B.SC.1, PENÉLOPE MAGAÑA, B.SC.2, ALEJANDRO OLVERA, M.SC.3, and ALEJANDRO ALAGÓN, PH.D.3

1Instituto Nacional de Producción de Biológicos A. N. L. I. S. “Dr. Carlos Gregorio Malbrán,” Ministerio de Salud y Ambiente, Buenos Aires, Argentina2Instituto Bioclón, Mexico City, Mexico3Instituto de Biotecnología, Universidad Autónoma de México, Cuernavaca, Morelos, Mexico

The toxic, biochemical, and immunological characteristics of L. boneti and L. reclusa venoms and its neutralization by anti–L. boneti andanti–L. reclusa antivenoms were studied. The electrophoretic profile showed very similar patterns and the toxic activities were very close.Immunological studies showed cross-reactivity among L. boneti and L. reclusa venoms, with L. boneti and L. reclusa experimentalantivenoms, and anti-L. gaucho and anti-L. laeta antivenoms. The venom of L. laeta showed low immunological reactivity with the NorthAmerican Loxosceles antivenoms. Experimental anti-North American Loxosceles antivenoms protected mice of the systemic toxicity andwere able to prevent necrosis in rabbit skin after the injection of the venom. Both antivenoms displayed cross neutralization. The resultsshowed that both Loxosceles venoms have very close toxic, biochemical, and immunological characteristics, and that either monospecificantivenoms or an antivenom raised with L. boneti and L. reclusa venoms as immunogens could be useful for treating bites by NorthAmerican Loxosceles spiders.

Keywords Spiders; Venom; Loxosceles; Antivenom; Therapeutics; Immunology; Neutralization; Toxicity

Introduction

Loxosceles spiders, known as “brown spider,” “violinist,”“killer,” “recluse,” “golden monk,” etc., are widely distrib-uted throughout the world. These spiders can be found inAfrica, America, Asia, and Europe (1), and from there wereintroduced in Australia (2). At least some of the 50 species ofLoxosceles can produce envenomation in humans, with verydifferent intensities ranging from a single lesion in the skin toacute renal failure and death (3,4). Together with Latrodec-tus, Atrax, Hadronyche, and Phoneutria spiders, Loxoscelesspiders are one of the few spiders whose bite can lead todeath and, together with Latrodectus (“black widow”), arethe two most dangerous spiders worldwide (3,5,6).

Loxosceles are reclusive, shy spiders that can be found inurban environments, including dwellings, and are oftenoverlooked due to their small size and sedentary behavior.The bite generally happens when the spider is accidentallypressed against the skin, while the victim is dressing or sleeping(2,5,7).

The most characteristic clinical sign of the Loxoscelesbite is a necrotic area at the site of the bite. This lesion isof remarkably large dimensions considering the small vol-ume of venom that this spider can inject: in the order oftenths of a microliter containing only a few micrograms ofprotein (8,9). Necrosis is followed by ulceration, whichmay take several months to heal. Local necrosis may beaccompanied by a mild systemic syndrome includingfever, malaise, itching, and exanthema. In addition,Loxosceles envenomation may produce a severe systemicsyndrome characterized by massive intravascular hemoly-sis and disseminated intravascular coagulation, sometimesaccompanied by thrombocytopenia and renal failure (3);the presence of myonecrosis was recently described (10).These alterations in the systemic syndrome can lead todeath (3,11).

It has been established that the main toxic componentresponsible for venom toxicity in Loxosceles envenoma-tion is a sphingomyelinase D (12–14). This enzyme leadsto local lesions by thrombus formation and migration ofwhite blood cells (15), and is responsible for intravascularhemolysis through alterations in the red blood cell mem-brane, since it causes activation of endogenous metallopro-teinases that cleave glycoproteins responsible for theinhibition of complement fixation by the alternative path-way (14). Several other enzymes have been described in

Address correspondence to Adolfo Rafael de Roodt, Área Inves-tigación y Desarrollo/Serpentario, I.N.P.B.-A.N.L.I.S. “Dr. C. G.Malbrán,” Av. Vélez Sarsfield 563, CP 1281, Buenos Aires,Argentina. E-mail: aderoodt@uolsinectis.com.ar

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Loxosceles venoms and antivenoms 679

Loxosceles venoms (6) and their participation in the patho-physiological events in envenomation are still being stud-ied. However, the pathophysiological mechanisms inLoxosceles envenomation have not yet been fully eluci-dated (16). For example, envenoming mechanisms mayvary to a large degree in the different species of experi-mental animals. This phenomenon can be easily recognizedin the very big differences in sensitivity to necrosis andlethality in mice, rats, and rabbits, even of different strainsor races (15,17,18).

In the United States, the spiders involved in human acci-dents are principally Loxosceles (L.) reclusa and, in secondplace, L. arizonica and L. desertica (19–22). There are sev-eral species of Loxosceles in Mexico, including L. reclusaand L. boneti. The latter can be found in Mexico in manylocalities of Guerrero, Puebla, and Morelos states (23), and itis the widest distributed species of this genus in Mexico (24).These spiders cause envenomation that may occasionally leadto death by systemic complications. In Mexico City duringthe period 1998–2002, five deaths by Loxosceles bite wereregistered (25).

Although it is a matter of discussion (26), the only spe-cific treatment for Loxosceles bites would be early admin-istration of antivenom (3,4). For this reason, we studiedsome toxic activities of venom gland homogenates ofL. reclusa and L. boneti in mice and rabbits and producedexperimental anti-L. boneti and anti-L. reclusa antivenomsin order to study their ability to neutralize the toxicity ofthe venoms in these animal species. In addition, some bio-chemical and immunochemical studies of the venoms andthe reactivity among different non-specific antivenoms tothese Loxosceles venoms and to their sphingomyelinaseswere made.

Materials and methods

Spider venom

L. boneti specimens were collected in Tuxpan and Iguala,Guerrero State, Mexico. L. reclusa spiders were collected inStillwater, Oklahoma, United States of America. Identifica-tion of the species was done by Dr. Norman I. Platnick of theAmerican Museum of Natural History, New York. Spiderswere kept in individual boxes, under controlled environ-mental conditions, having free access to filtered water untilextraction of the venom glands. Venomous apparatus weredissected and homogenized in 0.15 M NaCl with a Tenbro-ker tissue grinder for 5 min, as described previously(27,28). The homogenate was centrifuged for 5 min at 6000g, the supernatant was aspirated and 0.1 ml aliquots weredistributed in plastic tubes and kept at −20°C until use. Col-lection, identification and production of venom glandhomogenates of L. laeta from Lima, Peru, were done byDrs. Alfonso Zavaleta and Maria Salas-Arruz from thePeruvian University Cayetano Heredia.

Protein concentration was determined by the Bradfordmethod (29), using the Protein Assay Kit (Bio-Rad) andbovine seroalbumin (BioRad) as protein standard.

Sphingomyelinases D

Chromatographic fractions with sphyngomyelinase D activitywere isolated from L. boneti, L. reclusa, and L. laeta venomgland homogenates on a Sephadex G-75 (Pharmacia) columnexactly as described by Ramos-Cerrillo (24). The proteincontent of the chromatographic fractions with sphingomyeli-nase D activity was adjusted to a final concentration of 150μg/ml protein (Bradford Method, Protein Assay Kit, BioRad)in NaCl 0.15M.

Sodium Dodecyl Sulfate - Polyacrylamide gel Electrophoresis (SDS-PAGE)

Electrophoretic separation was performed on a vertical slabin a 12.5% acrylamide/bisacrylamide gel, using the discontin-uous system described by Laemmli (30) under reducingconditions (β-mercaptoethanol). Protein samples of thehomogenate supernatant (40 – 50 μg) were applied. Formolecular weight estimations, the gels were run with theBroad Range Kit of New England Bio-Labs Protein Marker(P7702S). The gels were fixed and stained with CoomasieBrilliant Blue R (Bio Rad).

Experimental animals

NIH, CD-1, or BALB/c mice (18–20 g) and Californian rab-bits (2.5–3.0 kg) were purchased from Harlan, S. A. de C.V., Mexico City, Mexico. CF-1 mice (18–20 g) and NewZealand rabbits were provided by the Instituto Nacional deProducción de Biológicos – A. N. L. I. S. “Dr. Carlos G.Malbrán,” Buenos Aires, Argentina (henceforth INPB). Theanimals were kept under controlled environmental condi-tions and received food and filtered water ad libitum.Ethical management of the animals used for all the assays,were those recommended by the National Research Councilof Mexico (31).

Determination of lethal potency

It was assessed by intraperitoneal (i.p.) injection in mice byconventional methods (32). CF-1, NIH, CD-1, and BALB/cmice (18–20 g, n=7 per dose level) were injected i.p. with dif-ferent doses of L. boneti venom gland homogenates in 0.5 mlof 0.15 M NaCl. In addition, BALB/c mice were challengedin the same manner with L. reclusa venom. The number ofsurviving mice 48 h after the injection was plotted as a functionof the dose of venom gland homogenate. The LD50 (definedas the amount of venom which kills 50% of the animals) wascalculated by the Spearman and Karber Method (33). In

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addition, BALB/c mice were injected subcutaneously withdoses of L. boneti venom gland homogenates and the lowestdose of venom killing 100% of injected mice, which was con-sidered to be the subcutaneous (s.c.) LD100, was recorded.

Determination of dermonecrotic activity

It was determined as described by Barbaro (17,18) withsome modifications. Briefly, different doses of the venom-ous gland homogenates were diluted with 0.15 M NaCl upto 100 μl (final volume) and injected intradermally in ashaved area of the dorsal skin of Californian or NewZealand rabbits. The animals were sacrificed 48 h afterinjection and the necrotic areas produced by the differentdoses of venom were determined. The necrotizing activitywas expressed as 1) minimal necrotizing dose (MND),which means the amount of venom that produced a necroticarea with mean diameters of 1 cm, or 2) as the homogenatedose which produced a 10 cm2 necrotic area, named NU(necrotizing unit). The reason for adopting this unit is thatthe conventional units for necrosis used in the study ofsnake venoms (34) give results with high variations whenthis venom is studied. This is because, in the model of rab-bit skin, small lesions (for example, 0.5 or 1 cm2) arepresent in the inferior low portion of the sigmoidal doseresponse curve. In contrast, the 10 cm2 area of necrosis is inthe logarithmic site of the dose-response curve.

Experimental antivenoms

The monovalent anti-L. reclusa antivenom (henceforth A-Lr)and the anti-L.boneti antivenom (henceforth A-Lb) were pre-pared as follows. Horses were immunized with six injectionsof 200 μg of venom gland homogenates every seven days.The first immunization was applied with Freund´s completeadjuvant and the second with Freund´s incomplete adjuvant.The subsequent immunizations were applied with venomgland homogenates diluted in 0.15 M NaCl.

On day 60, the horses were bled, after which the plasmaproteins were fractionated by precipitation with ammoniumsulfate and digested with pepsin by conventional techniques(35). The fractions containing the F(ab′)2 fragments wereextensively dialyzed against 0.15 M NaCl, adjusted to a finalprotein concentration of 35 ± 5 mg/ml, to which 0.25% phe-nol and 0.05 g thimerosal per liter were added as antimicro-bial agents. Both antivenoms were filtered through a 0.22 μmmembrane.

An experimental anti-Loxoscles laeta (A-Ll) antivenom pre-pared by conventional methods as previously described (38),was used for experiments of immunological cross reactivity.

Other antivenoms

For the immunodiffusion studies, antiarachnidic antivenomfrom the Butantan Institute (anti-L. gaucho), Sao Paulo,

Brazil (Batch 9509201, Exp. date September 1998, 100 ± 20mg protein/ml, henceforth A-Lg) and Anti-L. laeta monovalentantivenom (“Suero antiloxoscélico monovalente” – monova-lent anti-Loxosceles serum) from the Instituto Nacional deSalud, Peru (Batch 900258, Exp. Date 09–2000, 62 ± 7 mgprotein/ml) were used. For ELISA and neutralization assays tostudy the cross neutralization of heterologous anti-Loxoscelesantivenoms, A-Lg and the A-Ll (80 ± 20 mg/ml of protein con-tent) antivenoms were used. The A-Lg and the experimental A-Ll antivenoms were constituted by F(ab′)2 fragments of equineimmunoglobulins and the “monovalent anti-Loxosceles serum”was constituted by whole molecules of equine IgG.

ELISA

Anti Loxosceles antivenoms (A-Lb, A-Lr, A-Lg, and A-Ll)were titrated for their antibody activity using ELISA plates(Corning) coated with 1.0 μg / ml of venom gland homoge-nate as described by conventional classical methods (36).Coated plates were incubated 30 min at 37°C with the differ-ent antivenoms, or with normal horse immunoglobulin frac-tion or F(ab’)2 as controls. Goat Anti-Horse IgG peroxidaseconjugate (Sigma) was employed as a second antibody. Afterincubation of antivenom-Anti-horse IgG (30 min at 37°C),peroxidase reaction was started by the addition of ABTS(2,2’-Azino-bis(3ethylvenzotiazoline-6-sulfonic) acid) 0.1 Mcitrate buffer, pH 4.2, containing 0.03% hydrogen peroxide,and allowed to proceed for 15 min in the dark. The reactionwas stopped by the addition of 3.0 N sulfuric acid and read at405 nm using an ELISA (Labsystem Multiscan) reader.

Double immunodiffusion

This was performed on slides containing 5 ml of 1% Agarose(Sigma) in PBS pH 7.4 as described by Siles Villarroel withsome modifications (37). Wells were punched and filled with20 μl with a protein content of 150 μg/ml of sphingomyeli-nases of L. boneti, L. reclusa, or L. laeta or with 20 μl of a 1mg/ml solution of venomous gland homogenates (in 0.15 MNaCl) from each spider venom. The enzymes were con-fronted with different antivenoms (A-Lb, A-Lr, A-Lg, and“monovalent anti-Loxosceles serum”). After 48 h, the slideswere washed with 0.15 M NaCl for 72 h, changing the solu-tion each 12 h, dried at 37°C and stained with Amido Black.

Neutralization of lethal potency

Neutralization of lethal potency was determined in twodifferent ways, as follows.

Preincubation assay

A series of 6 BALB/c mice were injected i.p. with 3.0 i.p.LD50 of the supernatants of venom gland homogenates fromL. boneti or L. reclusa preincubated for 30 min at 37°C with

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either 0.15 M NaCl (positive controls), or different volumesof A-Lb or A-Lr antivenoms in a final volume of 0.5 ml. Sur-vival was recorded after 48 h. The neutralizing capacity wasexpressed as the effective dose (ED) 50% (ED50, i.e., the anti-venom dose which protects half of the injected mice after 48 h).Additionally, CF-1 mice were challenged with 3.0 i.p. LD50of L. boneti venom preincubated with different amounts ofA-Lb, A-Lr, A-Lg, or A-Ll antivenoms, as described above.The ED50s for each antivenom were also calculated.

Rescue assay

Groups of five BALB/c mice were injected with two s.c.(subcutaneous) LD100 of L. boneti venom contained in 0.2 mlof NaCl 0.15 M. After different intervals of time (30 to 300min), the mice were inoculated i.p. with different amounts ofA-Lb antivenom diluted in a final volume of 0.5 ml in NaCl0.15 M (50 to 500 μl). Survival was recorded 24 h after theinjection of the challenge dose. The results were expressed asthe percentage of surviving mice.

Neutralization of dermonecrotic activity

This was determined by two different methods, as follows.

Preincubation assay

Californian rabbits (n= 3 for each antivenom tested) were inoc-ulated intradermally as described with 20 μg of L. boneti (36MND or 7 NU) or L. reclusa (33 MND or 6.5 NU) venomgland homogenates preincubated with either 0.15 M NaCl(positive controls) or different doses of the A-Lb or A-Lr anti-venoms. The animals were sacrificed 48 h after injection andthe necrotic areas were measured in the dermal face of the skin.To test heterologous cross neutralization of necrosis by bothantivenoms, New Zealand rabbits (n= 2 for each antivenomtested) were challenged in the same way with 10 MND ofL. boneti venom preincubated with different doses of A-Lb, A-Lr, A-Lg, or A-Ll antivenoms using the same methodology.

The neutralizing capacity was expressed as ED50, whichmeans the dose of antivenom that reduced the necrotic-hemorrhagic area 50% when compared with the positive controls.

Rescue assay

Nine Californian rabbits were injected i.d. with 20 μg of L.boneti venom gland homogenate at three points (20 μg perpoint) on the back. After 60 or 150 min., groups of three rab-bits were injected intravenously with 2.5 ml of the Anti-L.boneti antivenom. After 48 h, the rabbits were sacrificed, thenecrotized areas were measured, and the percentage of inhibi-tion of the necrosis in the treated animals was estimated.

Statistics

Results are presented as mean ± standard deviation. In someinstances, those values were followed, in parenthesis, by the

95% confidence interval (CI). Lethal doses 50 (LD50) weredetermined by the Spearman and Karber method. Effectivedoses 50 (ED50), minimal necrotizing doses (MND), andELISA titers were estimated by nonlinear regression usingthe sigmoidal dose-response curve (variable slope) equationof the Prism–StatMate program (GraphPad Software, SanDiego, CA).

Results

The average protein content of the supernatant of the venomgland homogenate for L. boneti was 64 ± 15 μg (95% CI 34to 94 μg) per venom apparatus and 45 ± 7 μg (95% CI 31 to59 μg) per venomous apparatus of L. reclusa. This proteincontent is around 1/3 – 1/4 of that described in the venomousglands of L. laeta which possess around 180 μg of protein pervenomous apparatus (38,39).

SDS-PAGE of the homogenates showed protein bandswith molecular weights ranging from above 100 kDa to lessthan 20 kDa. Strong stained bands around the order of 66(corresponding to hemocyanin) and 30 kDa (corresponding tothe sphingomyelinases) were observed (Fig. 1).

The i.p. LD50 of L. boneti venom were 26 μg (95% CI 23to 28 μg) for CD-1 mice, 20 μg (95% CI 17 to 22 μg) for CF-1 mice, 19 μg (95% CI 13 to 26 μg) for NIH mice, and 17 μg(95% CI 13 to 19 μg) for BALB/c mice. The i.p. LD50 forL. reclusa venom gland homogenates in BALB/c mice was21 μg (95% CI 14 to 26 μg). The subcutaneous LD100 ofL. boneti venom in BALB/c mice was 40 μg.

Necrosis in the rabbit skin was produced with very smallamounts of venom. In Californian rabbits, the MND for L.boneti venom was 0.56 ± 0.50 μg and the NU 3.00 ± 1.50 μg,and in New Zealand rabbits the MND was 1.50 ± 1.00 μg andthe NU 4.48 ± 1.56 μg. For L. reclusa venom gland homoge-nates, the MND and the NU in Californian rabbits were 0.61± 0.50 μg and 3.10 ± 1.30 μg, respectively. Results were alsoexpressed as the calculated number of venomous apparatusescontaining the amount of venom neutralized by one milliliterof antivenom (Table 1).

Double immunoprecipitation experiments (Fig. 2) showedrecognition of the different anti-Loxosceles antivenoms(A-Lb, A-Lr, A-Lg, and “monovalent anti-loxoscelic serum”) onL. boneti, L. reclusa, and L. laeta venomous gland homoge-nates (Fig. 2A). Fractions with sphingomyelinase D activityfrom L. boneti and L. reclusa were recognized by all the anti-venoms (Fig. 2), whereas the fractions with sphingomyeli-nase D activity of L. laeta, venom was well recognized onlyby the homologous antivenom (“monovalent anti-Loxoscelesserum”) (Fig. 2B). These results are congruent with thoseobtained in the ELISA assays in which all the antivenomsrecognized L. boneti and L. reclusa venoms, although theNorth-American antivenoms (A-Lb and A-Lr) showed higherreactivity than that observed with the South Americanantivenoms (A-Ll and A-Lg) on both venoms (Fig. 3).

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The lethal potency of 3.0 LD50 of L. boneti venom glandhomogenate in BALB/c mice was neutralized by A-Lb withan ED50 of 35 μl (95% CI 33 to 37 μl) and an ED50 of 50 μl(95% CI 28 to 92 μl) by A-Lr (Table 1). The lethal potency of3.0 LD50 of L. reclusa venom gland homogenate was neutral-ized with an ED50 of 39 μl (95% CI 33 to 44 μl) of A-Lr andwith an ED50 of 41 μl (95% CI 37 to 46 μl) by A-Lb (Table1). When the experiments were performed with L. bonetivenom in CF-1 mice, the values of ED50s were 9 μl (95% CI7 to 10 μl) for A-Lb, 10 (95% CI 9 to 11 μl) for A-Lr, 15 μl(95% CI 12 to 18 μl) for A-Ll and 20 μl (95% CI 16 to 26 μl)for the A-Lg antivenom. The results of neutralization in CF-1mice are shown in Figure 4.

In the rescue experiments, we could observe protectionfrom lethality when the antivenom was applied no more than1h after the venom injection. Mice were protected from 2LD100 of L. boneti venom with doses of 350 μl of antivenomapplied 30 min (100% of survival) or 60 min (80% of sur-vival) after venom injection.

The experimental Anti–Loxosceles antivenoms were effec-tive in neutralizing the necrosis produced in the rabbit skin.The ED50 of A-Lb required to neutralize the necrosis pro-duced by 20 μg of L. boneti venom in Californian rabbits was6.3 μl (95% CI 4.8 to 8.3 μl). The ED50 of A-Lr on the neu-tralization of the necrosis produced by 20 μg of L. reclusavenom was 9.4 μl (95% CI 7.8 to 11.2 μl) (Table 1). Whenthe neutralization experiments on the necrotizing activity ofL. boneti venom were performed in New Zealand rabbits theED50 found were 8.0 μl (95% CI 6.8 to 9.3 μl) for A-Lr, 6.8 μl(95% CI 5.9 to 7.7 μl) for A-Lb, 8.9 (95% CI 8.8 to 9.0 μl) forA-Lg, and 21.5 (95% CI 14.9 to 30.9 μl) for A-Ll antivenoms(Fig. 4A).

In the rescue experiments, the necrotizing activity of L.boneti venom was inhibited by 72 ± 13% and 67 ± 11%,when the antivenom was inoculated 60 min or 150 min aftervenom injection, respectively.

Discussion

The venoms from L. boneti and L. reclusa showed high lethalpotency and necrotizing activity as described for the venom

Fig. 1. SDS-PAGE of venoms. Electrophoretic separation ofvenom gland homogenates was performed on a vertical slab of12.5% acrylamide under reducing condition. For molecularweight estimations, the gels were run with the Broad Range Kit ofNew England Bio-Labs Protein Marker (P7702S). Lane 1:molecular mass markers; Lane 2: L. boneti venom glandhomogenate (50 μg); and Lane 3: L. reclusa venom glandhomogenate (40 μg). The markers are indicated in the left andexpressed as kDa. The gels were fixed and stained with CoomasieBriliant Blue R (Bio Rad).

1 32

20.0

26.6

36.5

42.7

55.6

66.4

97.2

Table 1. Neutralization of L. boneti and L. reclusa venoms by Anti – L. reclusa (A-Lr) and Anti – L. boneti (A-Lb) antivenoms

Lethality in BALB/cmice Necrosis in Californian rabbits

Antivenoms L. reclusa venom L. boneti venom L.reclusa venom L. boneti venom

A – Lr 39 μl (33 to 44) [∼36 V.A./ml] 50 μl (28 to 92) [∼19 V.A./ml] 9 μl (8 to 11) [∼47 V.A./ml] 12 μl (8 to 14) [∼38 V.A./ml]A – Lb 41 μl (37 to 46) [∼34 V.A./ml] 35 μl (33 to 37) [∼27 V.A./ml] 10 μl (5 to 14) [∼30 V.A./ml] 6 μl (5 to 8) [∼50 V.A./ml]

Results are expressed in microliters as ED50 necessary to neutralize 3 LD50 of venom, as well as the calculated number of venomous apparatuses containingthe amount of venom neutralized by one milliliter of antivenom. The values correspond to the neutralization assays of lethality in BALB/c mice or neutral-ization of necrosis in Californian rabbits.V. A./ml: theoretical venomous apparatus neutralized by ml of antivenom. The 95% confidence intervals are expressed within parenthesis.

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Loxosceles venoms and antivenoms 683

of other species of North American and South AmericanLoxosceles (17,28,40). The differences in lethal potency inthe different strains of mice tested were not of the same mag-nitude as those described for the venom of L. intermediausing other different strains of mice (15); however, amongthe strains of mice tested BALB/c mice showed a slightlyhigher sensitivity to this venom. The necrotizing activity inrabbits was very similar in both races tested showing onlydifferences in the intensity of the edema, which was slightlyhigher in New Zealand Rabbits (data not shown).

The electrophoretic profile of venom gland homogenatesof both Loxosceles venoms (Fig. 1) showed strong stainedbands in the order of 60 kDa, in correspondence with themolecular mass of the hemocyanin (41) and around 30 kDa,in concordance with the molecular weights for the sphingo-myelinases (necrotizing factor) described in the venom ofthese spiders (24) and in the venom of South AmericanLoxosceles (12,13,42).

By double immunodiffusion we observed strong immu-nochemical cross-reactivity among venoms from L. bonetiand L. reclusa. Cross-reactivity was also found among theother Loxosceles venoms and antivenoms since A-Ll and A-Lg antivenoms produced faint bands of immunoprecipitationwith L. boneti and L. reclusa venoms (Figs. 2 and 3). Cross-reactivity and cross neutralization among North Americanand South American Loxosceles venom and antivenoms hasbeen recently described (43). The qualitative immunodiffusion

findings were consistent with the data generated by ELISAexperiments (Fig. 3); again A-Lb and A-Lr antivenoms gavehigher titers and overall response against L. boneti and L.reclusa venoms as compared with A-Ll and A-Lg antiven-oms. It is necessary to point out that the sphingomyelinasesof both North American Loxosceles venoms were well recog-nized by all the antivenoms studied, nevertheless theenzymes from L. laeta venom were only well recognized bythe homologous antivenom and to a lesser extent by the A-Lgantivenom (Fig. 2). These results agree with the amino acidsequence homologies between L. laeta sphingomyelinasesand other South American (44) and North American (24)sphyngomyelinases from Loxosceles venoms (Table 2) andwith the higher neutralization of L. laeta venom by homolo-gous antivenoms (38,45).

Immunological cross-reactivity was also supported by theresults obtained by experiments of cross-neutralization of thelethal and necrotizing activities. Neutralization of lethality inmice and/or necrosis in rabbits by heterologous anti-Loxosce-les antivenoms, has already been described for North andSouth American Loxosceles venoms (17,40,43). In past expe-riences, we found cross-reactivity and neutralizing capacityof L. boneti or L. laeta experimental antivenoms against thelethal and necrotizing activities of L. boneti or L. laetavenoms (46). In agreement with these previous results, in thepresent work we could observe interesting protectionprovided by South American Loxosceles antivenoms against

Fig. 2. A) Double immunodiffusion of the venomous gland homogenates of the L. boneti (Lb), L. reclusa (Lr), and L. laeta (Ll) venoms(concentration of each one 1 mg/ml, sample 20 μl), against anti L. boneti venom (A-Lb), anti L. reclusa venom (A-Lr), anti L. gauchovenom (Soro Antiaracnídico Polivalente, A-Lg), and anti L. laeta (Suero Antiloxoscélico, SA-Ll). B) Double immunodiffusion of thesphingomyelinases from L. boneti (S-Lb), L. reclusa (S-Lr), and L. laeta (S-Ll) venoms (concentration of each one 150 μg/ml, sample 20μl), against anti L. boneti venom (A-Lb), anti L. reclusa venom (A-Lr), anti L. gaucho venom (Soro Antiaracnídico Polivalente, A-Lg), andanti L. laeta venom (Suero Antiloxoscélico, SA-Ll).

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the necrotizing and lethal activities of L. boneti venom. How-ever, although A-Ll and A-Lg showed good neutralizingcapacity, it was not as potent as that observed using the NorthAmerican antivenoms A-Lb and A-Lr (Fig. 4), which showedhigher neutralizing potency against L. boneti venom (p < 0.05).These differences are greater if we consider that the proteincontent of the A-Lb and A-Lr antivenoms was at least half ofthat of the A-Lg or A-Ll antivenoms (Fig. 4).

Cross neutralization conferred by A-Lb and A-Lr antiven-oms against L. boneti or L. reclusa venoms was very high asthe ED50 s were very close for each antivenom (Table 1).

Some comments should be made with respect to the nomi-nal potency (that declared in the vial by the producer) of theantivenoms. When we compared the ED50 of A-Lb and A-Lragainst L. boneti venom, doses are very different dependingon whether the assay is performed using BALB/c or CF-1mice. The ED50 for the neutralization of 3 i.p. LD50 ofL. boneti venom was 35 μl for A-Lb (∼ 27 venomous apparatus

per milliliter of antivenom) in BALB/c mice and 9 μl (∼ 104venomous apparatus per milliliter) in CF-1 mice. Neutraliza-tion of this venom by A-Lr antivenom was achieved with anED50 of 50 μl using BALB/c mice (around 19 venomousapparatus per milliliter) or 10 μl in CF-1 mice (∼ 94 venom-ous apparatus per milliliter). These differences are moreimportant if the doses of venom required to reach 3.0 i.p.LD50 (around 60 μg for CF-1 mice and 51 μg for BALB/cmice) are considered. By the ED50 values, the susceptibilityof BALB/c mice is about four times higher than of CF-1mice, using the same pool of venom, the same batch of anti-venoms and the same technique for the assay. These differ-ences clearly indicate the complexity of the mechanismsinvolved in the toxicity of these venoms and the difficultiesin interpreting the results when different species or strains ofanimals are considered. These factors make it necessary toacknowledge the strain of mice or breed of rabbit used and itssusceptibility, and to test the potencies of the venom or itsneutralization by antivenoms in order to correctly interpretthe results from these types of assays.

The use of antivenom in Loxosceles envenomation is con-troversial. There are different opinions on this topic, in whichvery different points of view can be found and on whichsome authors have not a fully positive opinion (5,26,47,48).However, the use of antivenom is one of the very few thera-peutic tools available to treat these envenomations (3,5). Forexample, in South America the use of antivenom is stronglyrecommended and it is regularly used because of the clinicaleffectiveness observed in the early treatment of Loxoscelesenvenomation (3,4,7,16,49–51). Furthermore, health authori-ties from the countries with the largest numbers of accidentscaused by these spiders and experience in Loxosceles thera-peutics (Brazil and Peru) strongly recommend the use ofantivenom in the early stages of envenomation (3,52,53).

Experimentally, it was established that anti-Loxosceles mon-oclonal or polyclonal antibodies applied several hours afterexperimental envenomation are capable of neutralizing the tox-icity of the venom (54,55). In this work, we observed that theA-Lb antivenom showed neutralizing capacity against 2 LD100of L. boneti venom when applied one hour after injecting thevenom. In addition, this antivenom was able to neutralizenecrosis in rabbits by 35.7 MND (6.7 NU) of L. boneti venomwhen applied 2.5 hours after inoculation with the venom glandhomogenates. These results strengthen the possibility of thetherapeutic usefulness of this type of antivenom in the earlystages of envenomation by Loxosceles.

In the present work, we found high immunochemical reac-tivity and neutralizing ability using the antivenoms developedwith venom from geographically related Loxosceles. How-ever, the neutralization found using specific antivenoms, washigher than that observed with heterologous anti-Loxoscelesantivenoms (38,56). Recently, Tambourgi has described lowimmunochemical reactivity among recombinant sphingomy-elinases of L. intermedia an L. laeta (44) and it was suggestedthat for accidents involving L. laeta a specific serum therapycould be necessary (45).

Fig. 3. ELISA of Anti – L. laeta (A-Ll), Anti – L. gaucho (A-Lg),Anti – L. boneti (A-Lb), and Anti – L. reclusa (A-Lr) antivenomson L. boneti venom (3A) or L. reclusa venom (3B). The logarithmof the dilutions are indicated in the X axis and the absorbance at405 nm in the Y axis. (�) A-Lb, (�) A-Lr, (❍) A-Ll, and (�) A-Lg.OD: optical density.

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Fig. 4. Neutralization of the venom of L. boneti by different anti – Loxosceles antivenom. The bars express the ED50 required to neutralizethe necrotizing activity in New Zealand rabbits (4A) and the lethal activity in CF-1 mice (4B). The ED50s are expressed as the amount ofantivenom in microliters (a) or in mg of protein (b). A-Ll (anti –L. laeta venom), A-Lg (anti - L. gaucho venom), A-Lr (anti – L. reclusavenom), and A-Lb (anti – L. boneti venom).

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Table 2. Percentage of amino acid sequence identity between different isoforms of active sphyngomyelinases from different species ofLoxosceles

Sequence LlH-17 Ll-1 Ll-2 Ll-H13 Lr-1 Lb-1 Lr-2 La Li-P1 Li-D1 Li-P2

LlH-17 -Ll-1 99 -Ll-2 82 82 -Ll-H13 80 80 94 -Lr-1 60 59 60 60 -Lb-1 57 56 58 57 91 -Lr-2 60 59 61 61 90 84 -La 59 58 59 58 88 86 87 -Li-P1 60 59 59 59 84 82 85 81 -Li-D1 59 59 59 60 84 82 85 80 99 -Li-P2 59 58 59 60 83 82 87 80 90 90 -

There is a high degree of identity between L. boneti and L. reclusa sphingomyelinases (marked in bold). Also, the different percentage of identity amongthe enzymes from L. laeta and those from North American Loxosceles and other species of Loxosceles are given. The estimations of identities were madewith the program Protein Alignment (from the software GeneWorks 2.5.1, Intelligenetics, Inc.). Analyzed sequences were taken from ref. 58.Ll: L. laeta; L.r.: L. recluse; L.b.: L. boneti; L.a.: L. arizonica; and L.i.: L. intermedia. Numbers indicate the different isoforms of the sphyngomyelinases.

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However, cross-reactivity among Loxosceles venoms is avery useful fact for the production of antivenom and thetreatment of envenomation with heterologous Loxoscelesantivenoms (17,43). For example, the antivenoms currentlyused in South American countries are the anti-Arachnidicfrom Brazil (anti-L. gaucho) and “monovalent anti-Loxosceles serum” from Peru (anti-L. laeta) (11,38,57).This cross-reactivity among Loxosceles venoms permits theuse of heterologous antivenoms for the treatment ofLoxosceles bites in South American countries (3,17); never-theless, the availability of Loxosceles antivenoms is a bigproblem even in countries where this type of antivenom iswidely used (11). The inclusion of Loxosceles venoms, ortheir components with different characteristics in the immu-nogenic mixtures for antivenom production, could help toobtain good polyspecific Loxosceles antivenom whichcould yield better neutralization of the different Loxoscelesspiders’ venoms.

In conclusion, the cross-neutralization observed in thiswork using A-Lb and A-Lr antivenoms suggests that bothvenoms may be useful as immunogens for the developmentof an antivenom for treating envenomation by L. boneti and/or L. reclusa venoms. Also, it is probable that an antivenomwhich includes both venoms as immunogens or its sphingo-myelinases (24), as suggested for South American Loxosceles(44), could confer at least similar protection to that observedwith specific antivenoms, with a good coverage of the toxic-ity of North American Loxosceles venoms.

Acknowledgements

The authors greatly appreciate the collaboration of Dr. Nor-man I. Platnick from the American Museum of Natural His-tory, New York for the identification of the North AmericanLoxosceles spiders used in this work. The authors thank Dr.Alfonso Zavaleta-Martinez and Dr. Maria Salas-Arruz fromthe Peruvian University Cayetano Heredia for the provisionof venom gland homogenates from Loxosceles laeta venom-ous apparatus. We greatly appreciate the collaboration ofBlanca Ramos-Cerrillo in the purification of sphyngomyeli-nases from Loxosceles venoms and to Felipe Olvera for hiscollaboration in the laboratory work. We thank Dr. CarlosSevcik from the IVIC, Caracas, Venezuela for helping uswith the statistical interpretation of the results. We alsothank Dr. Charlotte Ownby for allowing us the use of herlaboratory for the extraction of L. reclusa venom.

References

1. Buchler W. Spiders. In: Buchler W, Buckley E, eds. Venomous animalsand their Venoms. Vol. III Venomous Invertebrates. New York: Aca-demic Press, 1971:197–277.

2. Isbister GK, White J. Clinical consequences of spider bites: Recentadvances in our understanding. Toxicon 2004; 43:477–492.

3. Ministerio de Saúde. Fundacaó Nacional de Saúde. In: Manual deDiagnóstico e Tratamento de Acidentes por Animais Peçonhentos. Minis-terio de Saúde, Fundação Nacional de Saúde, Brasilia, 1999:57–61.

4. Zavaleta Martínez-Vargas A. Loxoscelismo, un problema de salud en elPerú. Bol Of Sanit Panam 1987; 103:378–386.

5. White J, Cardoso JL, Fan HW. Clinical Toxicology of spiders’ bites. In:Meier J, White J, eds. Clinical Toxicology of Animal Venoms andPoisons. Boca Raton, FL: CRS Press, 1995:261–329.

6. da Silva PH, da Silveira RB, Appel MH, Mangili OC, Gremski W,Veiga SS. Brown spiders and Loxoscelism. Toxicon 2004; 44:693–709.

7. Siqueira Franca FO. Accidents by Loxosceles. Mem Inst Butantan1990; 52:63–64.

8. Babcock JL, Civello DJ, Geren CR. Purification and characterization ofa toxin from brown recluse spider (Loxosceles reclusa) venom glandextracts. Toxicon 1981; 19:677–689.

9. Smith CW, Micks DW. A comparative study of the venom and othercomponents of three species of Loxosceles. Am J Trop Med Hyg 1968;17:651–656.

10. Siqueira Franca FO, Barbaro KC, Abdulkader RCRM. Rhabdomyolisisin presumed viscerocutaneous loxocelism: report of two cases. Pirenop-olis, Brazil: VII Simposio da Sociedade Brasileira de Toxinologia,2002:130.

11. de Roodt AR, Salomón OD, Lloveras S, Orduna TA. Poisoning byLoxosceles spiders in Argentina. Medicina (Buenos Aires) 2001;62:83–94.

12. Tambourgi DV, Magnoli FC, Von Eickstedt VR, Benedetti ZC,Petricevich VL, da Silva WD. Incorporation of a 35-kilodalton purifiedprotein from Loxosceles intermedia spider venom transforms humanerythrocytes into activators of autologous complement alternative path-way. J Immunol 1995; 155:4459–4466.

13. Tambourgi DV, Magnoli FC, van den Berg CW, Morgan BP, de AraujoPS, Alves EW, Da Silva WD. Sphingomyelinases in the venom of thespider Loxosceles intermedia are responsible for both dermonecrosisand complement-dependent hemolysis. Biochem Biophys Res Commun1998; 251:366–373.

14. Tambourgi DV, Morgan BP, de Andrade MRG, Magnoli FC, van denBerg C. Loxosceles intermedia spider envenomation induces activationof an endogenous metalloproteinase resulting in cleavage of glycophor-ins from the erythrocyte surface and facilitating complement-mediatedlysis. Blood 2000; 95:683–691.

15. Tambourgi DV, Petrievich VL, Magnoli FC, Assaf SL, Jancar S, DiasDa Silva W. Endotoxemic-like shock induced by Loxosceles spidervenoms: Pathological changes and putative cytokine mediators. Toxi-con 1998; 36:391–403.

16. Barbaro KC, Costa Cardoso JL. Mecanismo de Acao do Veneno deLoxosceles e Aspectos Clínicos do Loxoscelismo. In: Costa Cardoso JL,De Siquiera Franca FO, Wen FH, Sant´ana Málaque CM, Hadad V Jr,eds. Animais Peconhentos no Brasil. Biología, Clínica e Terapéuticados Accidentes. Sao Paulo: Sarvier-FAPESP, 2003:160–174.

17. Barbaro KC, von Eickstedt VRD, Mota I. Antigenic cross-reactivity ofvenoms from medically important Loxosceles (Aranae) species inBrazil. Toxicon 1994; 32:687–693.

18. Barbaro KC, Ferreira ML, Cardoso DF, von Eickstedt VRD, Mota I.Identification and neutralization of biological activities in the venomsof Loxosceles spiders. Braz J Med Biol Res 1996; 29:1491–1497.

19. Escalante-Galindo P, Montoya-Cabrera MA, Terroba-Larios VM,Nava-Juarez AR, Escalante-Flores I. Local dermonecrotic loxoscelismin children bitten by the spider Loxosceles reclusa (the “violin” spider).Gac Med Mex. 1999; 135:423–426.

20. Sams HH, Hearth SB, Long LL, Wilson DC, Sanders DH, King LE Jr.Nineteen documented cases of Loxosceles reclusa envenomation. J AmAcad Dermatol 2001; 44:603–608.

21. Vetter RS, Barger DK. An infestation of 2,055 brown recluse spiders (Ara-neae: Sicariidae) and no envenomations in a Kansas home: implications forbite diagnoses in non endemic areas. J Med Entomol 2002; 39:948–951.

22. Wilson DC, King LE Jr. Spiders and spider bites. Dermatol Clin 1990;8:277–286.

Clin

ical

Tox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

UN

AM

on

10/1

8/11

For

pers

onal

use

onl

y.

Loxosceles venoms and antivenoms 687

23. Gertsch WJ, Ennik F. The spider Genus Loxosceles in North America,Central America and the West Indies (Araneae, Loxoscelidae). Bulletinof the American Museum of Natural History 1983; 175:264–360.

24. Ramos-Cerrillo B, Olvera A, Odell GV, Zamudio F, Paniagua-Solis J,Alagon A, Stock RP. Genetic and enzymatic characterization of sphin-gomyelinase D isoforms from the North American fiddleback spidersLoxosceles boneti and Loxosceles reclusa. Toxicon 2004; 44:507–514.

25. Sánchez-Villegas M del C, López de Silanes J. Use of a recombinantantivenoms for local loxoscelism. Acta Toxicológica Argentina 2005;13 (suppl.):11–12.

26. Isbister GK, Graudins A, White J, Warrell, D. Antivenom treatment inarachnidism. J Toxicol Clin Toxicol 2003; 41:291–300.

27. Babcok JL, Suber RL, Frith CH, Geren CR. Systemic effect in mice ofvenom apparatus extract and toxin from the brown recluse spider(Loxosceles reclusa). Toxicon 1981; 19:463–471.

28. Geren CR, Chan TK, Howell DR, Odell GV. Isolation and characteriza-tion of toxins from brown recluse spider venom (Loxosceles reclusa).Archives of Biochemistry and Biophysics 1976; 174:90–99.

29. Bradford MM. A rapid sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem 1976; 72:248–254.

30. Laemmli UK. Cleavage of structural proteins during the assembly ofthe head bacteriophage T4. Nature 1970; 227:680–685.

31. Comission of Life Sciences. Guía para el Cuidado y Uso de los Animales deLaboratorio. Lomelí C. ed. Mexican Academy of Sciences, Mexico, 2002.

32. Barretto Cicarelli RM, Siles Villarroel M, Zelante F. Avalicao da activ-idade toxica do veneno de Loxosceles gaucho em termos de DL50 e titu-lacáo do antiveneno em camundongos. Mem Inst Butantan 1983/84; 47/48:45–53.

33. World Health Organization. Progress in the Characterization of Ven-oms and Standarization of Antivenoms. Geneva: Offset Publication,WHO, 1981:23–24.

34. Theakston RDG, Reid HA. Development of simple standard assay pro-cedures for the characterization of snake venoms. Bulletin of the WorldHealth Organization 1983; 61:949–956.

35. Christensen PA. The preparation and purification of antivenin. Memo-rias do Instituto Butantan 1966; 22:245–250.

36. Theakston RDG, Lloyd-Jones MJ, Reid HA. Micro-ELISA for detectingand assaying snake venom and venom-antibody. Lancet 1977; 24:639–641.

37. Siles Villarroel MS, Zelante F, Furlanetto RS, Rolim Rosa R. Contribucaoao estudo imunoquímico de venenos botrópicos III. Analise dos compo-nentes antigénicos comuns a traves da dupla difusao e imunoelectroforeseen gel de agar. Mem Inst Butantan 1976/77; 40/41:241–250.

38. de Roodt AR, Litwin S, Dokmetjian JC, Vidal JC. A reduced immuni-zation scheme to obtain an experimental anti-Loxosceles laeta (“violin-ist spider”) venom. J Natural Toxins 2002; 11:193–203.

39. de Roodt AR, Gould I, Manzanelli VM. Corporal measures of Loxosce-les laeta and Kukulcania (Filistata) sp. from the province of BuenosAires. Acta Toxicológica Argentina 2005; 13 (suppl.):117–118.

40. Gómez HF, Miller MJ, Waggener MW, Lankford HA, Warren JS. Anti-genic cross-reactivity of venoms from medically important NorthAmerican Loxosceles spider species. Toxicon 2001; 39:817–824.

41. Estévez J, Dolab JA, Ramos-Cerrillo B, Olvera A, Paniagua-Solis JF,Magaña P, Litwin S, Salas M, Zavaleta A, Alagón A, de Roodt AR. Tox-icity and Immunological Reactivity of Two North American Loxosceles(“Brown Spider”) venoms. VIII Congresso da Sociedade Brasileira deToxinologia / VIII Symposium of the Pan American Section of the Inter-national Society on Toxinology, Angras dos Reis, 2004:19–23.

42. Barbaro KC. Biological, immunochemical and biochemical character-ization of venoms of spiders of the genus Loxosceles. Thesis. J VenomAnim Toxins 1997; 3:53.

43. Barbaro KC, Knysak I, Martins R, Hogan C, Winkel K. Enzymaticcharacterization, antigenic cross reactivity and neutralization of der-monecrotic activity of five Loxosceles spider venoms of medical impor-tance in the Americas. Toxicon 2005; 45:489–499.

44 Tambourgi DV, Pedrosa MM, Van Den Berg CW, Goncalves-De-AndradeRM, Ferracini M, Paixao-Cavalcante D, Morgan BP, Rushmere NK.Molecular cloning, expression, function and immunoreactivities ofmembers of a gene family of sphingomyelinases from Loxoscelesvenom glands. Mol Immunol 2004; 41:831–40.

45. de Oliveira KC, Goncalvez de Andrade RM, Piazza RM, Ferreira JM,van den Berg CW, Tambourgi DV. Variations in Loxosceles spidervenom composition and toxicity contribute to the severity of envenom-ation. Toxicon 2005; 45:421–429.

46. de Roodt AR, Estevez J, Litwin S, Dokmetjian JC, Paniagua J. Sometoxic and immunologic studies on Loxosceles venom gland homoge-nates from spiders of South America and North America. 6th Asia-Pacific Congress on Animal, Plant and Microbial Toxins and 11th

Annual Scientific Meeting of the Australasian College of Tropical Med-icine, Cairns, Australia, 2002:142.

47. Bravo M, Oviedo I, Farias P, Schenone H. Study of anti-loxoscelesserum action on hemolytic and ulcero-necrotic cutaneous effects ofLoxosceles laeta venom. Rev Med Chil. 1994; 122:625–629.

48. Schenone H, Saavedra T, Rojas A, Villarroel F. Loxoscelismo en Chile.Estudios epidemiológicos, clínicos y experimentales. Rev Inst MedTrop Sao Paulo 1989; 31: 403–415.

49. Martino O, Mathet H, Masini RD, Ibarra Grasso A, Thompson R, Gondell C,Bosch J. Emponzoñamiento humano provocado por venenos de origen ani-mal. Secretaría de Salud de la República Argentina, 1979:19–67.

50. Martino O, Orduna T. Patología cutánea ponzoñosa e infecciosa provocadapor agresiones de animales. Centro Municipal de Patologías RegionalesArgentinas (CEMPRA), Buenos Aires, 1995.

51. Torres JB, Marques M, Nicolella A, Severo Kluwe LH. Loxosceles. In:Nicolella A, Barros E, Torres JB, Marques M. Hospital de Clínicas dePorto Alegre, eds. Acidentes com Animais Peçonhentos. Consultarápida. Porto Alegre, 1997:127–129.

52. Zavaleta-Martínez Vargas A, Salas-Arruz M. Suero AntiloxoscélicoMonovalente Heterólogo (Equino). In: Zavaleta-Martínez Vargas A,Salas-Arruz M, Cabezas-Sánchez C, Chang-Neyra J, Carrillo-Parodi C.Farmacología de venenos y antivenenos de serpiente. Ministerio deSalud, Instituto Nacional de Salud Serie de Documentos N° 6, Lima,1998:117–121.

53. Sanabria H, Zavaleta A. Panorama epidemiológico del Loxoscelismo enel Perú. Revista de Medicina Experimental 1997; 14:33–40.

54. Gómez HF, Miller MJ, Warren JS. Intradermal anti-Loxosceles Fabfragments attenuate dermonecrotic arachnidism. Acad Emerg Med1999; 6:107–114.

55. Guilherme P, Fernandes I, Barbaro KC. Neutralization of dermone-crotic and lethal activities and differences among 32–35 kDa toxins ofmedically important Loxosceles spider venoms in Brazil revealed bymonoclonal antibodies. Toxicon 2001; 39:1333–1342.

56. Braz A, Minozzo J, Abreu JC, Gubert IC, Chávez–Olórtegui C. Devel-opment and evaluation of the neutralizing capacity of horse antivenomagainst the Brazilian spider Loxosceles intermedia. Toxicon 1999;37:1323–1328.

57. Grisolía CA, Stanchi NO, Francini F, Castro ES. Actividad de losCentros Antiponzoñosos. Primeros 20 años: 1975–1994. Provincia deBuenos Aires, Ministerio de Salud, 1996:33–37.

58. Ramos-Cerrillo BM. Producción, evaluación y caracterización de losinmunógenos recombinantes de las esfingomielinasas de L. boneti, L.reclusa y L. laeta. Master in Science Degree Thesis, Institute ofBiotechnology, Autonomous University of Mexico, 2005.

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