Produção de Soro Hiperimune para Potamotrygon motoro Müller & Henle, 1841 (Chondrichthyes – Potamotrygoninae): verificação da reação-cruzada frente às peçonhas de outras espécies de arraias e da neutralização das atividades edematogênica e miotóxica

Abstract

Stingray, belonging to the Potamotrygoninae subfamily are cartilaginous fish which are found in the watersheds of South America. These animals are provided with stingers at the base of the tail, suitable for their defense. The sting is covered by a tegument sheath containing mucous glands and venom. The victims often suffer poisonings by stingrays when they step on the animal's dorsum, hidden under the sand, on the edge of the beach. Consequently, the stingray moves tail abruptly and inserts the stinger on the victim, causing an extremely painful laceration, which usually leads to tissue necrosis. Accidents usually occur in distant and isolated places, usually without adequate medical care, so they are almost always not notified. There is no defined and effective therapy for these poisoning and health professionals not received adequate training to control this type of accident. The treatment is based on the use of analgesics, anti-inflammatories and antibiotics, as there is no specific antidote. Although medical procedures are able to control the clinical status of patients, the neutralization of toxins would be the ideal conduit to prevent the induction of signs and symptoms of poisoning by freshwater stingrays. In order to obtain an immunobiological able to neutralize the main effects induced by the poison, the present study aimed to analyze the neutralization, by hyperimmune sera, of the edematogenic and myotoxic activities induced by the poison of Potamotrygon motoro. Serums were obtained in Balb/c mice by means of intradermal immunization using either the dorsal extract or stinger extract of P. motoro adsorbed on aluminum hydroxide adjuvant. By Dot-ELISA and Western Blot analysis, it was possible to verify that the dorsal extract was as immunogenic as the stinger extract, inducing high titers of antibodies, which reacted with homologous (of the same species) and heterologous antigens (dorsum and stinger extracts from the species Paratrygon aiereba, Plesiotrygon iwamae, Potamotrygon orbignyi and Potamotrygon schroederi, all of freshwater), indicating that both types of extracts could be used in the production of antivenom to treat victims of poisoning by freshwater stingrays. From there, the power of the hyperimmune sera was verified in neutralizing, in vivo, the edematogenic and myotoxic activities induced by the stinger extract of P. motoro through two protocols: serum neutralization and vaccination. The first consisted of injecting the venom into the gastrocnemius muscle of Balb/c mice, and then administering either the antidorsal serum or the antistinger serum via ophthalmic venous plexus. The second one consisted in immunizing the mice with the dorsal or stinger extract adsorbed on aluminum hydroxide and challenging them with the venom of the stinger intramuscularly. The gastrocnemius were removed for histopathological and stereological analysis and blood was collected via the ophthalmic venous plexus for cytokine, CRP and CK dosing. Antidorsal and antistinger sera did not neutralize the edematogenic activity, but the serum neutralization and vaccination protocols partially neutralized the tissue damage induced by the stinger venom. Systemic rhabdomyolysis was only neutralized 100% in the animals vaccinated with the stinger extract. Cytokine analysis indicated that the serum neutralization protocol induced the release of cytokines from the Th1, Th2, Th17 and Treg profiles, while the vaccination protocol induced a Th1 response. The results indicate that the dorsal extract can be used as an alternative to the stinger extract (or together) in the production of immunobiologicals in treatments for freshwater poisoning.