Protein content of antivenoms and relationship with their immunochemical reactivity and neutralization assays.

CONTEXT: Therapy for snakebites relies on the application of antivenoms, which may be produced with different immunogenic mixtures of venom and possess different pharmaceutical characteristics. For these reasons, immunological cross-reactivity and heterologous neutralization were analyzed relative to the protein content of three antivenoms used in the Americas. METHODS: The antivenoms studied were composed of equine F(ab')2 fragments from animals immunized with Crotalinae venoms. The antivenoms were tested against venoms of seven pit viper species from Argentina, seven from Mexico, one from Costa Rica, and one from Colombia. RESULTS: Immunoblotting showed high cross-reactivity of all major protein bands with all the antivenoms tested. ELISA results also showed high cross-reactivity among the different venoms and antivenoms, and a high heterologous neutralization was observed. The results can be interpreted in different ways depending on whether the reactivity is considered in terms of the volume of antivenom used or by the amount of protein contained in this volume of antivenom. The antivenoms with high immunochemical reactivity and neutralizing capacity were those with higher protein content per vial; but when doses were adjusted by protein content, antivenoms of apparently lower neutralizing capacity and immunochemical reactivity showed at least similar potency and reactivity although volumetrically at higher doses. CONCLUSION: Protein content relative to neutralization potency of different products must be taken into account when antivenoms are compared, in addition to the volume required for therapeutic effect. These results show the importance of obtaining high-affinity and high-avidity antibodies to achieve good neutralization using low protein concentration and low-volume antivenoms.

Lymphatic route of transport and pharmacokinetics of Micrurus fulvius (coral snake) venom in sheep.

The contribution of the lymphatic system to the absorption and systemic bioavailability of Micrurus fulvius venom after subcutaneous (SC) administration was assessed using a central lymph-cannulated sheep model. Micrurus fulvius venom was administered either by intravenous bolus (IV) or subcutaneous injection (SC) in 12 sheep with and without thoracic duct cannulation and drainage. Venom concentration in serum and lymph was determined by a sandwich enzyme-linked immunosorbent assay (ELISA) in samples collected over a 6-hour period and in tissues harvested at the end of the experiment. Pharmacokinetic parameters were determined by a non-compartmental analysis. In the lymphatic cannulated group, over the 6 hours after the venom was administered, 69% of administered dose was accounted for in blood (45%) and lymph (25%). Negligible levels of venom were detected in organs and urine implying that the steady state observed after SC administration is maintained by a slow absorption process. Comparison of kinetics of the thoracic duct cannulated and non-cannulated groups showed that lymphatic absorption contributed in an important way to maintenance of this steady state. These results show that the limiting process in the pharmacokinetics of Micrurus fulvius venom following SC administration is absorption, and that the lymphatic system plays a key role in this process.

[Study on the venoms of the principal venomous snakes from French Guiana and the neutralization]. / Etude des venins des principaux serpents venimeux de Guyane française et de leur neutralisation.

We studied some biochemical, toxic and immunological characteristics of the venoms of Bothrops atrox, Bothrops brazili and Lachesis muta, Viperidae responsible for most of the bites of venomous snakes in French Guiana. Chromatographic (HPLC) and electrophoretical profiles (SDS-PAGE), lethal, hemorrhagic, defibrinogenating, coagulant, thrombin like, proteolytic, fibrino(geno)lytic and phospholipase activities were studied. In addition, the neutralization of some toxic activities conferred by four antivenins was compared. The chromatographic and electrophoretic profiles were different for the three venoms, showing differences between Bothrops and L. muta venoms. In general, bothropic venoms showed the highest toxic and enzymatic activities, while the venom of L. muta showed the lowest lethal, hemorrhagic and coagulant activities. The enzymes of bothropic venoms responsible for gelatinolytic activity were around 50-90 kDa. All the venoms were able to hydrolyze a and beta chains of the fibrinogen, showing different patterns of degradation. Although all the antivenoms tested were effective to various degrees in neutralizing the venom of B. brazili and B. atrox, neutralization of L. muta venom was significantly better achieved using the antivenom including this venom in its immunogenic mixture. For the neutralization of L. muta venom, homologous or polyvalent antivenoms that include the "bushmaster" venom in their immunogenic mixture should be preferred.

[Scorpions: spontaneous report in 2 reference centers in the city of Buenos Aires, 1997-2000]. / Escorpiones: denuncia espontánea en dos centros de referencia en la ciudad de Buenos Aires, 1997-2000.

From September 1997 to June 2000 148 scorpions (118 findings) from the province and city of Buenos Aires were sent to the INPB and the CeNDIE. The species involved were Tityus trivittatus (32/33 found in the city) and Bothriurus bonariensis (81/85 found in the province around the city). The population spontaneous report of both species increased from January 1999 to May 2000. The climatic, spatial and social variables that could be involved in these phenomena were analyzed. The influence of the media was essential for the 'peak' generation. However, the amount of findings or accident by scorpions, as well as the area of the city colonized by T. trivittatus, have shown a progressive increase during the last decades.

A study on the experimental envenomation in mice with the venom of Tityus trivitattus Kraepelin 1898 (Scorpiones, Buthidae) captured in Argentina.

Although Tityus trivitattus is the only scorpion species reported to cause severe human envenomation in Argentina, no previous studies on its venom have been done. Telson homogenates from T. trivitattus specimens collected in Santiago del Estero, Cordoba, and Buenos Aires were employed to study their protein composition and toxicity to mice. Regardless of the site of collection, electrophoretic analysis showed bands at 205, 150, 100, 40, 32, and 13 kDa or smaller. FPLC gel filtration showed three major peaks and 6-8 minor peaks with similar elution volumes. One of the minor peaks from FPLC containing a component of approximately 8 kDa was lethal to mice. Mice injected intravenously with different doses of homogenates presented severe autonomic signs like tachypnea, tachycardia, sialorrhea, lacrimation, profuse sweating, diarrhea, dyspnea, and death. Pathology studies of lungs showed severe congestion of alveolar capillaries, pulmonary edema, and hemorrhagic areas. The kidneys showed glomerular as well as tubular lesions and exocrine glands showed areas of necrosis. The calculated LD50 was 0.38 +/- 0.08 telsons per 20 g mouse, which suggests a lethal potency similar to that of T. serrulatus venom. The lethal potency of 5.0 LD50 of T. trivitattus telson homogenate was neutralized by both an anti-T. trivitattus and a heterologous anti Tityus with ED50 values of 41 +/- 19 and 170 +/- 42 microl, respectively.

Escorpiones: denuncia espontánea en dos centros de referencia en la ciudad de Buenos Aires, 1997-2000. / [Scorpions: spontaneous report in 2 reference centers in the city of Buenos Aires, 1997-2000]

From September 1997 to June 2000 148 scorpions (118 findings) from the province and city of Buenos Aires were sent to the INPB and the CeNDIE. The species involved were Tityus trivittatus (32/33 found in the city) and Bothriurus bonariensis (81/85 found in the province around the city). The population spontaneous report of both species increased from January 1999 to May 2000. The climatic, spatial and social variables that could be involved in these phenomena were analyzed. The influence of the media was essential for the peak generation. However, the amount of findings or accident by scorpions, as well as the area of the city colonized by T. trivittatus, have shown a progressive increase during the last decades.

A comparison of different methods to assess the hemorrhagic activity of Bothrops venoms.

The hemorrhagic activity of Bothrops (B.) alternatus, B. ammodytoides, B. jararaca, B. jararacussu, B. moojeni and B. neuwiedii venoms from specimens captured in Argentina was assayed after i.d. injection to mice. The hemorrhagic haloes produced by each venom had different color intensities, although no significant differences were observed by measurement of the average diameters or the weight of the excised hemorrhagic haloes. Conversely, important differences were found by measuring the amount of hemoglobin extracted from excised hemorrhagic haloes of similar size produced by different venoms. The relationship between the amount of hemoglobin extracted and the weight of the excised hemorrhagic haloes was linear, with a slope (hemoglobin released per gram of hemorrhagic halo) characteristic for each venom, and proportional to the potency. On this basis, the activity of B. alternatus, B. ammodytoides and B. jararaca is similar, about 1.5 times higher than that of B. jararacussu and B. moojeni venoms and threefold higher than that of B. neuwiedii venom. Thus, measurement of the of hemoglobin released provides additional information in comparative studies, and may be used to assess the antihemorrhagic potency of antivenoms.

Some toxic and enzymatic activities of Bothrops ammodytoides (yarará ñata) venom.

Bothrops ammodytoides, the smallest representative of this genus, is found only in Argentina. Venom was extracted from thirty adult specimens (35-70 cm in length, 90-300 g in weight) captured in the Province of Buenos Aires and kept in captivity. Venom yield was 3-30 mg. SDS-PAGE showed strong bands at 14.0; 23-25; 45; 54 and 63 kDa and weak bands at 17.0; 30.0; 40.0 and 85.0 kDa. Toxic activities were: LD50 (intravenous, mice) 0.5+/-0.2 microg/g; minimal procoagulant dose on human plasma (MPD-P) 35+/-2 mg/l; and minimal defibrinogenating dose (MDD, mice) 6-12 microg. Hemorrhagic and/or necrotic activities appear to play a major role in lethality; minimal hemorrhagic dose (MHD, mice) is 10+/-2 microg/g and minimal necrotizing dose (MND, mice) is 38+/-5 microg. The LD50, MPD-P and MND are among the lowest in venoms from Bothrops species found in Argentina. B. ammodytoides venom exhibited high proteolytic and phospholipase A2 activities. Most of the B. ammodytoides venom components cross-react with Bivalent Bothropic antivenom (Instituto Nacional de Producción de Biológicos ANLIS Dr. G. Malbrin, against B. alternatus and B. neuwiedii venoms). One ml of antivenom neutralizes 1.2 mg of B. ammodytoides venom.

[Accidents due to lepidoptera with special reference to Lonomia sp]. / Accidentes por lepidópteros con especial referencia a Lonomia sp.

Human poisoning by Lepidoptera (butterflies, caterpillars and moths) has been known to physicians since old times. In the last few years, several factors have been causative of a great number of contacts between different stages of these insects and humans, occasionally with epidemic features. Species of medical interest in Argentina are those related to Megalopygidae, Saturniidae, Hemileucidae, Arctiidae, Notodentidae and Eucliedae families. Among all the species involved, it is important to describe the pathology produced by contact with Lonomia obliqua caterpillar (Saturniidae). The caterpillar Lonomia has several enzymes with procoagulant and fibrinolytic activities, acting on various components of the hemostatic system inducing hemorrhage that can lead to death. In the South of Brazil during the last years caterpillars of this butterfly caused a great number of cases of erucism including some deaths. In the past two years, there have been some cases of poisoning by Lonomia obliqua in Misiones, in the North East of Argentina on the border with Southern Brazil. This presents a potential risk, taking into account the poor knowledge regarding this insect and its venom in Argentina. This brief actualization gives some biochemical, clinical and epidemiologic tools towards understanding human Lepidopterism, an uncommon medical problem in this country.

Hematological values of some Bothrops species (Ophidia - Crotalidae) in captivity

Blood samples of 50 healthy specimens from each of the following species: Bothrops alternatus, Bothrops jararacussu, Bothrops moojeni, and Bothrops neuwiedi diporus all kept in captivity were taken to determine the hematocrit (PCV) value, red blood cell count (RBC), total leukocyte (WBC) and differential leukocyte count, thrombocyte count, mean corpuscular volume (MCV), hemoglobin concentration (HbC), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). These hematological parameters were compared to those obtained from other Bothrops species. PCV values, RBC, hemoglobin, WBC count, and differential leukocyte count are within the range of values reported for other Bothrops species, while the thrombocyte count was significantly lower. All the hematological parameters obtained from the four studied Bothrops species were higher than those described for B. ammodytoides.

Accidentes por lepidópteros con especial referencia a Lonomia sp. / [Accidents due to lepidoptera with special reference to Lonomia sp]

Human poisoning by Lepidoptera (butterflies, caterpillars and moths) has been known to physicians since old times. In the last few years, several factors have been causative of a great number of contacts between different stages of these insects and humans, occasionally with epidemic features. Species of medical interest in Argentina are those related to Megalopygidae, Saturniidae, Hemileucidae, Arctiidae, Notodentidae and Eucliedae families. Among all the species involved, it is important to describe the pathology produced by contact with Lonomia obliqua caterpillar (Saturniidae). The caterpillar Lonomia has several enzymes with procoagulant and fibrinolytic activities, acting on various components of the hemostatic system inducing hemorrhage that can lead to death. In the South of Brazil during the last years caterpillars of this butterfly caused a great number of cases of erucism including some deaths. In the past two years, there have been some cases of poisoning by Lonomia obliqua in Misiones, in the North East of Argentina on the border with Southern Brazil. This presents a potential risk, taking into account the poor knowledge regarding this insect and its venom in Argentina. This brief actualization gives some biochemical, clinical and epidemiologic tools towards understanding human Lepidopterism, an uncommon medical problem in this country.

[Cross neutralization of bothrops jararacussu venom by heterologous antivenoms]. / Neutralización cruzada de veneno de Bothrops jararacussu por sueros antiofidicos heterologos.

We have studied the immunochemical cross-reactivity and cross-neutralization of the lethal potency, hemorrhagic, necrotizing, procoagulant and (indirect) hemolytic activities of Bothrops jararacussu venom by the standard antivenoms produced in Argentina. These antivenoms are horse immunoglobulin F (ab')2 fragments from animals immunized with 1) Crotalus durissus terrificus venom (Monovalent Anticrotalic antivenom); 2) Bothrops alternatus and B. neuwiedii venoms (Bivalent Botropic antivenom); 3) B. alternatus, B. neuwiedii, B. jararaca and B. jararacussu venoms (Tetravalent Bothropic, or "Misiones" antivenom) and 4) B. alternatus, B. neuwiedii and C. d. terrificus venoms (Trivalent Botropic-Crotalic antivenom). In preincubation experiments, all the heterologous antivenoms neutralized the toxic and biological activities of B. jararacussu venom with a potency at least as high as the Tetravalent Botropic (i.e. the only homologous) antivenom, in which B. jararacussu venom was included as immunogen. These results suggest the possibility of using heterologous antibothropic antivenoms for the treatment of snake bites by B. jararacussu.

Neutralización cruzada de veneno de Bothrops jararacussu por sueros antiofidicos heterologos. / [Cross neutralization of bothrops jararacussu venom by heterologous antivenoms]

We have studied the immunochemical cross-reactivity and cross-neutralization of the lethal potency, hemorrhagic, necrotizing, procoagulant and (indirect) hemolytic activities of Bothrops jararacussu venom by the standard antivenoms produced in Argentina. These antivenoms are horse immunoglobulin F (ab)2 fragments from animals immunized with 1) Crotalus durissus terrificus venom (Monovalent Anticrotalic antivenom); 2) Bothrops alternatus and B. neuwiedii venoms (Bivalent Botropic antivenom); 3) B. alternatus, B. neuwiedii, B. jararaca and B. jararacussu venoms (Tetravalent Bothropic, or [quot ]Misiones[quot ] antivenom) and 4) B. alternatus, B. neuwiedii and C. d. terrificus venoms (Trivalent Botropic-Crotalic antivenom). In preincubation experiments, all the heterologous antivenoms neutralized the toxic and biological activities of B. jararacussu venom with a potency at least as high as the Tetravalent Botropic (i.e. the only homologous) antivenom, in which B. jararacussu venom was included as immunogen. These results suggest the possibility of using heterologous antibothropic antivenoms for the treatment of snake bites by B. jararacussu.

A study on the venom yield of venomous snake species from Argentina.

A study on the venom yield of snakes from Argentina over a three year period was carried out on adult specimens of Bothrops alternatus (n = 74); Bothrops neuwiedii (n = 127); Bothrops ammodytoides (n = 30); Bothrops moojeni (n = 14); Bothrops jararaca (n = 14); B. jararacussu (n = 6); Crotalus durissus terrificus (n = 120) and Micrurus spp. (n = 6) as well as with 12 specimens of newborn C. d. terrificus kept in captivity. While for each species there was a positive correlation between venom yield and number of snakes milked, the correlation with the snake's body weights after individual milkings was even better, suggesting that the size of the snakes is more important in determining the venom yield than the number of snakes milked or the specimen's sex. Individual milkings indicated that, in addition to the snake size, when the amount of venom is normalized per 100 g body weight there is a species specific difference in venom yield. It follows the order B. jararacussu > B. moojeni approximately = B. jararaca approximately = B. alternatus > B. neuwiedii> Micrurus spp approximately = B. ammodytoides> C. d. terrificus. Although the venom yield per 100 g body weight of newborn C. d. terrificus specimens is 2-fold higher than that of adults, no correlation was observed between venom yield and body weight.

Cross-reactivity and heterologous neutralization of crotaline antivenoms used in Argentina.

The immunochemical cross-reactivity and neutralizing capacity of four crotalinae antivenoms consisting in equine F(ab')2 fragments and available in Argentina (bothropic Bivalent, against Bothrops alternatus and B. neuwiedii venoms; bothropic Tetravalent, against B. alternatus, B. neuwiedii; B. jararaca and B. jararacussu venoms; bothropic crotalic Trivalent, against B. alternatus, B. neuwiedii and Crotalus (C.) durissus terrificus venoms and anticrotalic against C. d. terrificus venom) were studied against B. alternatus, B. ammodytoides; B. jararaca; B. jararacussu, B. moojeni; B. neuwiedii and C. d. terrificus venoms. SDS-PAGE analysis of the Bothrops venoms showed protein bands of high (>40 kDa) medium (20-40 kDa) and low (<15 kDa) molecular weights, while that of C. d. terrificus exhibited a large amount of material with molecular weight of 15.0 kDa or lower. Immunoblotting showed a high cross-reactivity of all the major protein bands with all the antivenoms (even heterologous) tested. All the antivenoms were effective in neutralizing the lethal activity of the venoms tested, and in some cases (B. jararaca and B. jararacussu) heterologous antivenoms exhibited similar neutralizing capacity than the homologous ones. In spite of the differences in biochemical composition and pharmacology, Bothropic antivenoms displayed a significant neutralizing capacity on lethal activity of C. d. terrificus venom. In addition, all the antivenoms (including the anticrotalic) were highly effective in neutralizing the hemorragic, necrotizing, procoagulant, and proteolytic activities. The antivenoms tested produced different degrees of inhibition of phospholipase A2 activity, which exhibited a certain specificity but was also related to the enzyme content in the venom.

[Neutralizing capacity of ++antiophidic sera against the venom of Bothrops moojeni (lance-headed viper)]. / Capacidad neutralizante de los sueros antiofídicos frente al veneno de Bothrops moojeni (caisaca, lanzadera).

In the production of current therapeutic antisera used in Argentina for Bothrops snakebite, the Bothrops moojeni venom is not used as immunogen, since this snake is not considered a serious public health problem. Accidents caused by this species have not been reported in this country even though Bothrops moojeni is not unfrequent in some regions of Misiones. Despite the high degree of immunological cross reactivity found among the Crotalinae venoms and, in this particular case, among the venoms from the Bothrops Genus, there exists a significant intraspecific variation in venom composition, particularly in specimens arising from different geographic regions. In this study, the antivenoms prepared at the Instituto Nacional de Producción de Biológicos A.N.L.I.S. Dr. Carlos G. Malbrán have been tested for their immunochemical cross reactivity and neutralizing ability of enzymatic and toxic activities of venom from Argentinian specimens of Bothrops moojeni from Misiones. Immunological cross-reactivity was tested by double-immunoprecipitation, immunoelectrophoresis, Western-blot and ELISA. Neutralizing ability of antivenoms against proteolytic, indirect hemolytic activity, procoagulant activity, he-morrhagic activity and necrotizing activity. The Lethal Dose 50 was 1.5 mg/kg body weight; this value is located in the range to those obtained with the venom from Brazilian specimens. It was observed that all the antivenoms exhibited a strong immunochemical cross reactivity and that they were able to neutralize in different degree both, enzymatic and toxic activities of B. moojeni venom. From these results, it can be assumed that the antivenom tested could be employed successfully in cases of B. moojeni snakebites.

[A case of human sparganosis in Argentina]. / Un caso de esparganosis humana en Argentina.

A male patient from Peru presented a nodule localized in the left costal region. All other clinical and laboratory data were normal. Upon biopsy, an helminth parasite emerged from the subcutaneous tissue, which presented a marked eosinophil infiltrate. The helminth was classified as a plerocercoid larva of Spirometra; the species was not determined. Since Spirometra are not common in Argentina, it is presumed that the patient was infested during his two year sojourn in the peruvian forest. Some epidemiological aspects are discussed. As far as we know, this is the first case of subcutaneous sparganosis and the second of sparganosis reported in Argentina.