Luis Cerdas

An alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica

The ability of several batches of polyvalent antivenom to neutralize indirect hemolytic activity of Bothrops asper venom was studied using a sensitive plate test. All samples of antivenom tested effectively neutralized this activity. A highly significant correlation was observed between neutralization of indirect hemolysis and neutralization of lethal activity. This simple and sensitive in vitro test could be used to monitor antibody levels in horses immunized to produce polyvalent antivenom.

Neutralization of proteolytic and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom

The polyvalent antivenom produced at the Instituto Clodomiro Picado, Costa Rica, was tested for its capacity to neutralize proteolytic and hemorrhagic activities of ten Costa Rican crotaline venoms. In experiments with preincubation of venom and antivenom, the latter efficiently neutralized proteolytic activities of nine venoms, with ED50 ranging from 50 to 300 microliters antivenom/mg venom. The venom of Bothrops nummifer was neutralized less efficiently (ED50 = 760 microliters/mg.) Antivenom was also very effective in neutralizing hemorrhagic activity, having its lowest neutralizing ability against the venom of B. picadoi (ED = 430 microliters/mg) and its highest towards the venom of B. asper (Pacific region) (ED50 = 47 microliters/mg). There was a significant correlation between the ability of antivenom to neutralize proteolytic and hemorrhagic effects. In spite of the ability of antivenom to neutralize hemorrhage when incubated with venom prior to injection, hemorrhage was only partially neutralized when antivenom was administered i.v. at different time periods after envenomation. This suggests that the rapid development of local hemorrhage, instead of the absence of antivenom antibodies, is the explanation for the poor neutralization observed in these types of experiments.

Comparative study on coagulant, defibrinating, fibrinolytic and fibrinogenolytic activities of Costa Rican crotaline snake venoms and their neutralization by a polyvalent antivenom.

The coagulant, defibrinating, fibrino lytic and fibrinogenolytic activities of venoms from ten species of Costa Rican crotaline snakes were studied, together with the neutralization of these effects by a polyvalent antivenom. The venoms of Bothrops asper, B. schlegelii, B. nummifer, B. godmani, Lachesis muta and Crotalus durissus induced a coagulant effect in vitro, and all of them, with the exception of B. nummifer, also induced defibrination in vivo. The four non-coagulant venoms (B. lateralis, B. ophryomegas, B. nasuta and B. picadoi) induced a degradation of the alpha (A) chain of fibrinogen, thereby inhibiting coagulation. However, they did not induce defibrination upon i.v. injection. All of the venoms showed fibrinolytic activity in vitro. Polyvalent antivenom was effective in the neutralization of coagulant, defibrinating, fibrinolytic and fibrinogenolytic activities of these venoms, with the exception of coagulant effect induced by C. durissus venom. Since only three venoms are used in the immunization of horses, these results demonstrate the high degree of immunological cross reactivity between components affecting coagulation in Costa Rican crotaline snake venoms.

Neutralizacion de los efectos locales del veneno de Bothrops asper por un antiveneno polivalente

Abstract Neutralization of lethality, myonecrosis, hemorrhage and edema induced by Bothrops asper venom in mice was studied using the polyvalent antivenom produced in the Instituto Clodomiro Picado. The neutralizing effect ( ed 50 ) on each of these toxic activities varied; the neutralization of lethal and hemorrhagic effects being more effective than the neutralization of myonecrosis and edema. With independent inoculation of venom and antivenom, antivenom was not effective in neutralizing edema-forming activity. The myonecrotic effect was only partially neutralized when serum was given i.v. immediately after envenomation; however, antivenin effectively neutralized the hemorrhagic activity. The ineffectiveness of antivenom in neutralizing edema and myonecrosis could be partially explained by the rapid development of these effects. Hence, the time interval between envenomation and antivenom administration and the route of serum administration both play an important role in the neutralization of local effects.

Pharmacological activities of a toxic phospholipase A isolated from the venom of the snake Bothrops asper

A toxic phospholipase A was isolated from the venom of Bothrops asper. It induced skeletal muscle damage, anticoagulant effects and edema in the foot pad. The toxin had an intravenous LD50 of 95 micrograms/16-18 g mouse body wt and an intraventricular LD50 of 0.42 micrograms/16-18 g mouse body wt. Upon intramuscular and intravenous injections, the toxin induced a prominent increase in serum creatine kinase (CK) levels; only the CK-MM isozyme increased markedly. The toxin induced CK and creatine release from skeletal muscle incubated in vitro. The rate of efflux of creatine was higher than that of CK, although both markers were partially released as early as 15 min after incubation. The toxin also induced elevation of serum levels of lactic dehydrogenase isozymes. However, histological examination of skeletal muscle, kidneys, heart and lungs revealed cell damage only in skeletal muscle. The toxin was not cytotoxic to erythrocytes, lymphocytes or macrophages. In addition, it did not induce a mitogenic response on lymphocytes. In the absence of albumin in the medium, there was no significant difference between myotoxic activities in Ca2+-free and Ca2+-containing bathing solutions. However, when albumin was added, there was a significantly higher myotoxic effect in the presence of Ca2+. Thus, although phospholipolytic activity of the toxin plays a role in muscle damage when albumin is present, the toxin induces muscle damage even when phospholipase A activity is inhibited.

Comparative study of the edema-forming activity of costa rican snake venoms and its neutralization by a polyvalent antivenom

The edema-forming activity of eight Costa Rican crotaline snake venoms and its neutralization by a polyvalent antivenom were studied using the mouse footpad test. All of the venoms induced edema, the highest activity being present in the venoms of Bothrops lateralis and Bothrops picadoi. When experiments were performed with preincubation of venom and antivenom, neutralization of edema was poor. Moreover, it was observed that, with some venoms, edema increased when large doses of antivenom were used. This effect was also observed when some venoms were incubated with coral snake antivenom, suggesting that venoms may release some pharmacologically active component(s) from antivenom, since the latter contains traces of alpha-2 and beta globulins. Based on these findings, an alternative approach to the study of the neutralization of edema was used; in this new method, antivenom was injected i.v. before venom administration, thereby avoiding preincubation. With this technique, a much better neutralization of edema was observed, although with some venoms it was still poor. Venoms contain low molecular weight factors which induce edema, suggesting that lack of immunogenicity of some components may cause a poor neutralization. However, such components are responsible for only a minor portion of the edema induced by crude venoms. It is suggested that experiments in which venom and antivenom are preincubated preincubated in testing the neutralization of edema should be avoided, and that a more adequate approach may be an independent inoculation of venom and antivenom.

Local effects induced by coral snake venoms: Evidence of myonecrosis after experimental inoculations of venoms from five species

The local effects induced by intramuscular inoculations of venoms from six species of coral snakes were studied in mice. Venoms of Micrurus nigrocinctus nigrocinctus, M. n. mosquitensis, M. alleni, M. frontalis, M. carinicauda and M. surinamensis induced prominent myonecrosis which was observed histologically. From a morphological point of view all these venoms induced a similar pattern of myonecrosis, characterized by a conspicuous alteration of the intracellular structure. This myotoxic activity was corroborated by an increase in plasma creatine kinase levels 3 hr after i.m. injection of M. n. nigrocinctus, M. n. mosquitensis, M. frontalis and M. carinicauda venoms. M. mipartitus venom did not induce myonecrosis. None of the venoms induced edema or hemorrhage at the site of injection.

Ability of a polyvalent antivenom to neutralize the venom of Lachesis muta melanocephala, a new Costa Rican subspecies of the bushmaster

Several toxic and enzymatic activities of the venom of L. m. melanocephala were studied. This venom has many similarities with that of L. m. stenophrys, although there are quantitative differences in venom activities, as well as in the immunodiffusion patterns of these venoms when reacted against polyvalent antivenom. This antivenom was tested for its ability to neutralize a series of toxic and enzymatic effects of L. m. melanocephala venom. A new method to study myonecrosis, based on the quantitation of residual creatine kinase in injected muscle, was used. Antivenom was highly effective in neutralizing lethal, hemorrhagic, myotoxic, edema-forming, defibrinating, caseinolytic and fibrinolytic activities when venom and antivenom were incubated prior to the test or, in the case of edema-forming activity, when antivenom was administered before venom injection. On the other hand, when antivenom was injected i.v. at different time intervals after venom injection neutralization of lethality was good, although neutralization of local effects, i.e. hemorrhage and edema, was poor. These results indicate that polyvalent antivenom contains antibodies capable of neutralizing toxic and enzymatic activities of L. m. melanocephala venom. Moreover, the partial inability of antivenom to neutralize local effects when administered after venom injection is probably due to the rapid development of these effects once venom is injected.

Skeletal muscle necrosis induced by a phospholipase A2 isolated from the venom of the coral snake Micrurus nigrocinctus nigrocinctus

1. The venom of the coral snake Micrurus nigrocinctus nigrocinctus was fractionated by reverse-phase high performance liquid chromatography and an acidic myotoxic phospholipase A2 was purified to homogeneity. 2. After intramuscular injection, the toxin induced rapid and drastic myonecrosis, as serum creatine kinase levels increased markedly, reaching their highest values by 1.5 hr. 3. Ultrastructural observations indicate that the plasma membrane was the first structure to be affected, with the presence of focal disruptions in its integrity. 4. Myofilaments were hypercontracted and formed dense clumps. Sarcoplasmic reticulum integrity was lost, as evidenced by the presence of many small vesicles in the cellular space. 5. Some mitochondria were swollen, whereas others contained dense intracristal spaces and flocculent densities. Moreover, some had only one membrane. 6. In conclusion, pathogenesis of myonecrosis induced by this phospholipase A2 is similar to that induced by crude Micrurus nigrocinctus nigrocinctus venom.

Estudio comparativo de los venenos de serpiente Cascabel (Crotalus durissus durissus) de ejemplares adultos y recien nacidos

Venoms from adult and newborn Central American rattlesnakes (Crotalus durissus durissus) were compared for lethal, proteolytic, hemorrhagic, myonecrotic, edematigenous and in vitro hemolytic activities. Electrophoretic and immunoelectrophoretic patterns showed some differences between these venoms. Venom from newborn snakes was devoid of hemorrhagic and edematigenous activities, whereas the venom from adult specimens induced these effects. On the other hand, newborn snake venom showed higher lethality and indirect hemolytic activity, and lower proteolytic activity, than venom from adult specimens. Both types of venoms induced only slight myonecrosis in mice, as judged by histological observation. The ED50 of an antivenom, in terms of absolute weight neutralized per ml of serum, was lower for the newborn specimens venom than for adults venom, however, for each venom the number of LD50 neutralized was similar.