Search

Antivenom production against Loxosceles venom relies on horses being immunized and bled for plasma harvest. One horse can partake in several cycles of antivenom production, which will require years of constant venom and adjuvant inoculation and bleeding. The actual impact on the health of horses that participate in several antivenom-producing cycles is unknown. Therefore, this study aimed to evaluate the general health status of horses that underwent at least six cycles of loxoscelic antivenom production. Seven crossbred horses that had partaken in six to eight complete antivenom-producing cycles were used and established as the immunized group (IG). Under the same handling and general management, eleven horses were established as the control group (CG). The horses were evaluated regarding their general clinical status and had their blood sampled, and an ECG recorded. The IG presented lower RBC and PCV, despite keeping values within inferior limits for the species. Renal function was not impaired, and liver-related enzymes were higher than those in the CG, probably due to liver exertion from immunoglobulin synthesis. ECG showed some abnormalities in the IG, such as atrioventricular block and a wandering atrial pacemaker, corroborated by an increase in CK-MB. The cardiovascular abnormalities were mainly found in the horses that participated in several antivenom-producing cycles. The overall results indicate that these horses had some impairment of their general health status. Once available, some alternative, less toxic antigens should replace the venom for immunization of horses used for antivenom production.

Loxoscelism is the clinical condition triggered after the bite of spiders of the genus Loxosceles. The main species involved in accidents in South America are L. intermedia, L. laeta, and L. gaucho. The only specific treatment is the anti-Loxosceles serum produced with crude venoms. As phospholipases D (PLDs) trigger most of the effects observed in accidents, we developed and evaluated second-generation sera using mutated PLDs as antigens. Three isoforms of PLDs with site-directed mutations without biological activities were used for rabbit immunizations: D32A-E34A (L. gaucho), W230A (L. intermedia), and H12A-H47A (L. laeta). Sera were produced using crude venoms of three species of Loxosceles enriched with mutated recombinant PLDs (MIX) or using only mutated PLDs (REC). Immunizations stimulated the immune system from the second immunization with higher antibody production in the REC group. In vivo neutralization assays demonstrated that both sera reduced edema and dermonecrosis caused by Loxosceles intermedia crude venom. Follow-up of animals during the immunization protocols and in the neutralization assays demonstrated that the mutated proteins and the sera are safe. Results demonstrate the potential of using mutated recombinant PLDs in total or partial replacement of Loxosceles venoms in animal immunizations to produce anti-Loxosceles sera for treatments of Loxoscelism.

Bites of brown spiders (Loxosceles spp.) are responsible for dermonecrotic lesions and potentially systemic envenoming that can lead to death. The only effective therapy is the use of the antivenom, usually produced in horses. However, little is known about the consequences of the systematic use of the Loxosceles venom and adjuvants and of the bleedings on antivenom-producing horses. Thus, the aim of this study was to evaluate the clinical changes in horses in their first immunization protocol for Loxosceles antivenom production. Eleven healthy horses, never immunized, were evaluated in three different periods: T0 (before immunization); T1 (after their first venom immunization); and T2 (after their first bleeding). Horses were clinically evaluated, sampled for blood, and underwent electrocardiographic (ECG) recordings. Several suppurated subcutaneous abscesses occurred due to the use of Freund’s adjuvants and thrombophlebitis due to systematic venipunctures for the bleeding procedures. ECG showed arrhythmias in few horses in T2, such as an increase in T and R waves. In summary, the immunization protocol impacted on horses’ health, especially after bleeding for antivenom procurement.

Phospholipases-D (PLDs) found in Loxosceles spiders’ venoms are responsible for the dermonecrosis triggered by envenomation. PLDs can also induce other local and systemic effects, such as massive inflammatory response, edema, and hemolysis. Recombinant PLDs reproduce all of the deleterious effects induced by Loxosceles whole venoms. Herein, wild type and mutant PLDs of two species involved in accidents—L. gaucho and L. laeta—were recombinantly expressed and characterized. The mutations are related to amino acid residues relevant for catalysis (H12-H47), magnesium ion coordination (E32-D34) and binding to phospholipid substrates (Y228 and Y228-Y229-W230). Circular dichroism and structural data demonstrated that the mutant isoforms did not undergo significant structural changes. Immunoassays showed that mutant PLDs exhibit conserved epitopes and kept their antigenic properties despite the mutations. Both in vitro (sphingomyelinase activity and hemolysis) and in vivo (capillary permeability, dermonecrotic activity, and histopathological analysis) assays showed that the PLDs with mutations H12-H47, E32-D34, and Y228-Y229-W230 displayed only residual activities. Results indicate that these mutant toxins are suitable for use as antigens to obtain neutralizing antisera with enhanced properties since they will be based on the most deleterious toxins in the venom and without causing severe harmful effects to the animals in which these sera are produced.

Spiders of the genus Loxosceles, popularly known as Brown spiders, are considered a serious public health issue, especially in regions of hot or temperate climates, such as parts of North and South America. Although the venoms of these arachnids are complex in molecular composition, often containing proteins with distinct biochemical characteristics, the literature has primarily described a family of toxins, the Phospholipases-D (PLDs), which are highly conserved in all Loxosceles species. PLDs trigger most of the major clinical symptoms of loxoscelism i.e., dermonecrosis, thrombocytopenia, hemolysis, and acute renal failure. The key role played by PLDs in the symptomatology of loxoscelism was first described 40 years ago, when researches purified a hemolytic toxin that cleaved sphingomyelin and generated choline, and was referred to as a Sphingomyelinase-D, which was subsequently changed to Phospholipase-D when it was demonstrated that the enzyme also cleaved other cellular phospholipids. In this review, we present the information gleaned over the last 40 years about PLDs from Loxosceles venoms especially with regard to the production and characterization of recombinant isoforms. The history of obtaining these toxins is discussed, as well as their molecular organization and mechanisms of interaction with their substrates. We will address cellular biology aspects of these toxins and how they can be used in the development of drugs to address inflammatory processes and loxoscelism. Present and future aspects of loxoscelism diagnosis will be discussed, as well as their biotechnological applications and actions expected for the future in this field.