Identification and neutralization of biological activities in the venoms of Loxosceles spiders

The biological activities of the venom of three species of spiders of the genus Loxosceles were studied (L. gaucho, L. laeta and L. intermedia). The dermonecrotic and lethal activities are shared by all three Loxosceles venoms. Only low levels of proteolytic, myotoxic and phospholipase A2 activities were demonstrable even when a large amount of venom was used. No direct hemolytic activity was detected. L. intermedia venom was the most lethal (LD50 0.48 mg/kg), the L. laeta venom was the least lethal (LD50 1.45 mg/kg) whereas L. gaucho venom showed an intermediate value (LD50 0.74 mg/kg). The anti-Loxosceles serum used (anti-arachnidic serum) was able to neutralize the most important activities (i.e., dermonecrotic and lethal activities) of the three venoms. SDS-PAGE and immunoblotting using the anti-arachnidic serum showed that almost all venom antigens were recognized by this antiserum. The possible mechanisms of action of the Loxosceles venom are discussed.     

Immunoglobulin G and F(ab')2 polyvalent antivenoms do not differ in their ability to neutralize hemorrhage, edema and myonecrosis induced by Bothrops asper (terciopelo) snake venom

The ability of whole immunoglobulin G (IgG) and F(ab')2 polyvalent (Crotalinae) antivenoms to neutralize the hemorrhagic, edema-forming and myotoxic activities of Bothrops asper venom was studied. Both antivenoms were adjusted to the same neutralizing potency against lethal and hemorrhagic activities in experiments where venom and antivenoms were incubated before injection. Thus, in these experimental conditions, differences in the neutralizing ability in experiments involving independent injection of venom and antivenoms would depend mainly on the different pharmacokinetic profiles of whole IgG and F(ab')2 antivenoms. Experiments involving local injection of venom followed by intravenous administration of antivenom at either 0, 15 or 30 min after envenomation did not reveal any significant difference between whole IgG and F(ab')2 products concerning neutralization of hemorrhage, edema and myonecrosis induced by B. asper venom. The three effects were neutralized by antivenoms only to a partial extent and neutralization decreased as the time-lapse between envenomation and antivenom administration increased. Moreover, with the exception of one time-interval, no significant differences in the neutralization of hemorrhage were observed when antivenom was administered by the intramuscular or intraperitoneal route. The results do not support the assumption that F(ab')2 antivenom is more effective than whole IgG antivenom in the neutralization of local hemorrhage, edema and myonecrosis induced by B. asper venom in mice.     

Purification and characterization of prophenoloxidases from pupae of Drosophila melanogaster

The effects of heparin and other polyanions on the myotoxicity of Bothrops jararacussu venom and purified bothropstoxin (BthTX) were investigated. The release rate of creatine kinase (CK) from isolated extensor digitorum longus muscle and the plasma CK activity of mice were used to quantify the results. The myotoxic effects of B. jararacussu venom or BthTX were inhibited by preincubation of these agents with one of the following: a heterogeneous heparin preparation (designated 'heparin'), low mol. wt heparin (H-4500) or dextran sulfates (DS-8000 and DS-500,000). Non-sulfated dextran (D-40,000) and two chondroitin sulfates were ineffective. The antimyotoxic effects of the polyanions are ascribed to their forming inactive acid-base complexes with the basic myotoxins of Bothrops venoms. Gel-filtration experiments in Sephadex provided direct evidence for complex formation between heparin and BthTX. Intravenous (i.v.) administration of H-4500 or DS-8000 opposed the increase in plasma CK activity induced by a subsequent i.m. injection of venom or BthTX. In contrast, pretreatment with i.v. heparin or DS-500,000 enhanced the venom-induced increase in plasma CK activity. This effect was not observed (1) when the animals were treated with a polyvalent antivenom, which inhibits the coagulation and local stasis induced by Bothrops venoms, and (2) when BthTX, which has no thrombotic or hemorrhagic properties, was the myotoxic agent. The potentiation of the venom-induced increase in plasma CK activity by heparin and DS-500,000 is ascribed to improved washout of the CK released from damaged fibers, because of the anticoagulant properties of the drugs.     

Inhibition of hemorrhagic activities of various snake venoms by purified antihemorrhagic factor obtained from Japanese Habu snake

The ability of purified antihemorrhagic factor isolated from the serum of Japanese Habu (Trimeresurus flavoviridis) was tested on 17 snake venoms to inhibit their hemorrhagic activity. The factor strongly inhibited that of the venoms of Crotalus horridus horridus and Vipera latastei gaditana in addition to that of the homologous (T. flavoviridis) venom. Hemorrhagic activity of Agkistrodon halys blomhoffi, Agkistrodon contortrix contortrix, Bothrops atrox asper and Crotalus atrox venoms was also remarkably inhibited, but that of Vipera mauretanica to less extent. The hemorrhagic activities of nine other snake venoms were not inhibited.     

Ability of wedelolactone, heparin, and para-bromophenacyl bromide to antagonize the myotoxic effects of two crotaline venoms and their PLA2 myotoxins

We examined the ability of wedelolactone, heparin and para-bromophenacyl bromide to antagonize the myotoxic activity in mice of venoms from Crotalus viridis viridis and Agkistrodon contortrix laticinctus and two phospholipase A2 myotoxins, CVV myotoxin and ACL myotoxin, isolated from them. Myotoxicity was measured by the increase in plasma creatine kinase (CK) activity at two hours and histological changes in extensor digitorum longus muscle (EDL) at three hours after injection of the test solution. Both heparin and wedelolactone independently reduced the myotoxic effect of both crude venoms and both myotoxins, but wedelolactone was more effective. Wedelolactone plus heparin reduced the myotoxic effect of CVV myotoxin more than either antagonist alone. The PLA2 inhibitor, para-bromophenacyl bromide (pBPB), reduced the myotoxic effect of both myotoxins more than either wedelolactone or heparin. On the other hand, the myotoxic effect of polylysine was not reduced by either wedelolactone or para-bromophenacyl bromide, but it was reduced by heparin. These results indicate that wedelolactone, para-bromophenacyl bromide and heparin are antagonists of these two phospholipase A2 myotoxins, and that antagonism by the first two compounds may be due to a more specific interaction with these proteins than that by the latter.     

Development of snake antivenom antibodies in chickens and their purification from yolk

Adult white leghorn hens hyperimmunised with Brazilian snake venoms of the genus Bothrops and/or Crotalus produced antibodies capable of recognising, combining with and neutralising the toxic and lethal components of the venoms. The antibodies were first detected by an enzyme-linked immunosorbent assay two weeks after starting the immunisation schedule, reached the highest titres by the third week and remained high for at least 24 weeks. These antibodies are transferred to the egg yolk from which they were isolated as enriched IgY preparations by a combination of methods using positive and negative precipitation with sodium sulphate and/or caprylic acid. The yolk-derived IgY preparations contained antibodies which blocked the phospholipase A2-dependent haemolytic activity of both venoms and the haemorrhagic activity of Bothrops venom, and neutralised the toxic lethal activities of the venoms with good efficacy. The median effective dose (ED50) of the IgY anti-Bothrops venom was 592.5 microliters/2LD50 and, 1.0 ml neutralised 0.0675 mg of venom. The ED50 of the IgY anti-Crotalus venom was 457.5 microliters/3LD50 and 1.0 ml neutralised 0.075 mg of venom.     

Detection and isolation of bovine immunodeficiency-like virus (BIV) in dairy herds of Costa Rica

Serological (Western blot) detection of bovine immunodeficiency-like virus (BIV) in Holstein dairy herds is reported in Costa Rica for the first time, as well as the isolation of the virus, from a seropositive bovine, by cocultivation of peripheral blood mononuclear cells (PBMC) with embryonic rabbit epithelial (EREp) cells. The isolated strain, BIVCR1, reacted similarly in Western blot as the reference strain BIV R29 and is clearly distinguishable from bovine leukaemia virus (BLV). The data suggest an association between BIV infections and BLV infections, as it has been reported elsewhere. From these results it can be concluded that BIV is present in Costa Rica and it is suggested that these viral infections will probably follow the epidemiological parameters of BLV infections in Costa Rica, reaching high infection rates in dairy herds.     

Venom variability among several Tityus serrulatus specimens

Individual differences in venom composition among several Tityus serrulatus specimens collected in the same area were examined by enzyme-linked immunosorbent assay (ELISA). Polyclonal antibodies raised against whole venom and against the alpha-type (toxin IV-5 or Ts IV) and the beta-type toxin (toxin gamma or Ts VII) were used to study specific variations in the venom. The ELISA results indicated clear differences among the scorpion venoms examined. The lethality (LD50) determined by subcutaneous injections of pooled venom with the same characteristics showed an interesting correlation between the expression level of each component studied and the lethal effect of the venom. Among the groups analysed, the group with the highest concentration of alpha-type toxin showed the highest toxicity. The groups with the lowest level of toxicity were those with a low concentration of alpha-type toxin. The results show that the lethality of the venom varies from specimen to specimen and suggest that alpha-type toxin must be the major lethal component in the whole venom.     

Dynamics of the Lens culinaris agglutinin-lactotransferrin and serotransferrin complexes, followed by fluorescence intensity quenching of fluorescein (FITC) with iodide and temperature

Envenomation by Thalassophryne nattereri fishes are an important medical problem in northeast of Brazil, causing in human victims considerable pain and edema followed by necrosis. Venom obtained from fresh captured specimens of this fish was tested in vitro or in animal models for a better characterization of its toxic activities. Intradermal injection of the venom in the foot pad of mice induced local edema and hemorrhage followed a few hours later by necrosis. Subcutaneous injection of the venom induced systemic effects consisting in jerking motions, paralysis of hind limbs, erection of hair, rotational movements and violent convulsions followed by death. Dead animals showed hyperemia of the small intestine and lungs. The venom showed distinct edematous, necrotizing and hemolytic activities, a low level of hemorrhagic, myotoxic and proteolytic activities and no detectable phospholipase A2 activity. SDS-PAGE analysis of the crude venom showed at least 17 components with the major band located around Mw = 19,000. Almost all proteins stained by amido black were also revealed by Western blotting with antibodies to T. nattereri venom. Fractionation of the venom by either gel filtration or cation exchange chromatography resulted in a few distinct peaks but in both situations the biological activities were located in only one of the peaks which corresponded to basic proteins with approximately Mw = 47,000. Heating of the venom at 56 degrees C for 60 min completely destroyed its biological activities. All venom toxic activities except edema were completely neutralized after in vitro incubation with anti-T. nattereri serum.     

Phlebotomines (Diptera: Psychodidae) collected at a Costa Rican dairy farm in a vesicular stomatitis endemic area

This study determined the species composition of sand flies in a vesicular stomatitis endemic area, at Carrizal, Alajuela, Costa Rica. Eight species were collected as follows: Lutzomyia volcanensis (Fairchild & Hertig), L. shannoni (Dyar), Lu. pia (Fairchild & Hertig), L. sanguinaria (Fairchild & Hertig), L. youngi Feliciangeli & Murillo, L. longipalpis (Lutz & Neiva), L. serrana (Damasceno & Arouck), and L. gomezi (Nitzulescu).     

The detection of hemorrhagic proteins in snake venoms using monoclonal antibodies against Virginia opossum (Didelphis virginiana) serum

Most snakes and a few warm-blooded animals have a resistance to snake venoms because of naturally occurring antihemorrhagins found in their sera. The antihemorrhagins in serum of Virginia opossum (Didelphis virginiana) neutralize hemorrhagic activity by binding to hemorrhagins in snake venoms. The binding characteristic of antihemorrhagins in D. virginiana serum was used to develop a five-step western blot. The detection of hemorrhagic proteins were measured indirectly with antihemorrhagins in Virginia opossum serum and with DV-2LD#2, a monoclonal antibody specific for Virginia opossum antihemorrhagins. Snake venoms were separated by native-PAGE, transferred to a Millipore Immobilon-P membrane and then incubated with crude Virginia opossum serum. The hemorrhagins in snake venom bind to antihemorrhagins in Virginia opossum serum which react with DV-2LD#2 a monoclonal antibody that is specific for Virginia opossum antihemorrhagins. DV-2LD#2 monoclonal antibody inhibits antihemorrhagic activity in Virginia opossum serum when mixed in equal amounts. The inhibition of antihemorrhagins by DV-2LD#2 monoclonal antibody suggests specificity. DV-2LD#2 monoclonal antibody does not recognize antihemorrhagins in gray woodrat (Neotoma micropus) serum. The five-step western blot reveals two well-defined bands which represent hemorrhagins found in Western diamondback rattlesnake (Crotalus atrox) venom. Venoms from 15 different snake species were examined to determine the usefulness of the five-step western blot. Other hemorrhagic venoms (Great Basin rattlesnake (C. viridis lutosus), Prairie rattlesnake (C. viridis viridis), Tancitaran dusky rattlesnake (C. pusillus), Northern Mojave rattlesnake (C. scutulatus scutulatus type B) and Northern Pacific rattlesnake (C. v. oreganus)) had one single band in the five-step western blot. DV-2LD#2 did not bind to the non-hemorrhagic venoms and reacted with 50% of the hemorrhagic venoms used in this study. The monoclonal antibody, CAH, reacted with all the hemorrhagic venoms except for the venom of the King cobra (Ophiophagus hannah) and did not react with the non-hemorrhagic venoms. The hemorrhagic binding site of CAH monoclonal antibody and the antihemorrhagin in Virginia opossum are different binding sites. The five-step western blot will be a very useful assay for determining hemorrhagic activity without using live animals.     

Proteins isolated from the venom of the common tiger snake (Notechis scutatus scutatus) promote hypotension and hemorrhage

Notechis scutatus scutatus venom contains several toxic acidic proteins called HTa-i which promote hypotension and hemorrhage in mice. They have apparent mol. wts in the 18,000-21,000 range, i.v. LD50 values between 0.5 and 1.5 micrograms/g, and no detectable phospholipase, arginine esterase, proteolytic or hemolytic activities. A polyclonal antibody raised against HTg binds to other purified proteins, suggesting that they are isoforms of the same protein. Many other elapid crude venoms contain proteins which recognize the polyclonal antibody raised against HTg. Crotalid and viperid crude venoms do not recognize this antibody, although some of their component proteins are known to exhibit hypotensive and hemorrhagic activities. A combination of gel-filtration on Sephacryl S-200, cation-exchange and anion-exchange chromatography allows isolation of the N. s. scutatus proteins in high purity. They are the first hypotension-inducing proteins to be purified from an Australian elapid.     

Global variations in cancer survival. Study Group on Cancer Survival in Developing Countries

Population-based cancer registries from Algeria, China, Costa Rica, Cuba, India, the Philippines, and Thailand are collaborating with the International Agency for Research on Cancer in a study of cancer survival in developing countries. Comparisons with the SEER program results of the National Cancer Institute in the United States, and the EUROCARE study of survival in European countries revealed considerable differences in the survival of patients with certain tumors associated with intensive chemotherapeutic treatment regimes (Hodgkin's disease and testicular tumors), more modest differences in the survival of patients with tumors for which early diagnosis and treatment confer an improved prognosis (carcinomas of the large bowel, breast, and cervix), and only slight differences for tumors associated with poor prognosis (carcinomas of the stomach, pancreas, and lung). With limited resources to meet the challenge of the increasing incidence of cancer expected in the next few decades, health authorities in developing countries should be aware of the importance of investing in a range of cancer control activities, including primary prevention and early detection programs as well as treatment.     

Hemorrhagic and edema-forming activity and histologic changes in the mouse footpad induced by venoms from Argentinian Bothrops and Crotalus genuses

Hemorrhagic, oedema-forming activities and histopathological alterations in the mouse footpad induced by Bothrops and Crotalus snake venoms from Argentina. Hemorrhagic and oedema-forming activities of various Bothrops and Crotalus snake venoms from Argentina were studied, together with histological alterations in the mouse footpad. The highest oedema-forming activity was found in the venom of B. jararaca, followed by B. jararacussu, B. neuwiedii diporus, B. alternatus, and Crotalus durissus terrificus. Regarding hemorrhage, the highest activity was found in the venom of B. neuwiedii diporus, followed by B. jararacussu, B. alternatus, and B. jararaca. No hemorrhage was observed after injection of Crotalus durissus terrificus venom, in agreement with histological observations of injected footpads. Histological analysis revealed a conspicuous inflammatory reaction in the injected footpad, characterized by oedema and an inflammatory infiltrate rich in polymorphonuclear leucocytes. Necrotic blood vessels and dilated lymphatic vessels were observed after injection of B. jararaca and B. jararacussu venoms, and myonecrosis was evident in tissue of mice injected with B. alternatus and B. neuwiedii diporus.     

Common themes and surprising differences in small G-proteins

In Costa Rica, an annual outbreak of infant diarrheal disease (December and January) was reported since 1976, and rotavirus was incriminated later as the main etiological agent (1976-1981). Apparently the disease has not been systematically studied in Costa Rica after 1981. For that reason the occurrence of the outbreak was retrospectively documented for 1993-1995 and etiology was studied in 48 children treated for diarrhea at the Nacional Children Hospital (capital city of San Jose) during December, 1994 and January, 1995. Rotavirus (33%) and coronavirus (27%) were the main agents. To our knowledge, this is the first time that these viruses are incriminated in an outbreak of diarrhea.     

Molecular basis for prokaryotic specificity of magainin-induced lysis

Magainins and mastoparans are examples of peptide antibiotics and peptide venoms, respectively. They have been grouped together as class L amphipathic helixes [Segrest, J.P., et al. (1990) Proteins 8, 103-117] because of similarities in the distribution of Lys residues along the polar face of the helix. Class L venoms lyse both eukaryotic and prokaryotic cells whereas class L antibiotics specifically lyse bacteria. The structural basis for the specificity of class L antibiotics is not well understood. Sequence analysis showed that class L antibiotics have a Glu residue on the nonpolar face of the amphipathic helix; this is absent from class L venoms. We synthesized three model class L peptides with or without Glu on the nonpolar face: 18LMG (LGSIWKFIKAFVGGIKKF), [E14]18LMG and [G5,E14]18LMG. Hemolysis, bacteriolysis, and bacteriostasis studies using these peptides showed that the specificity of lysis is due to both the presence of a Glu residue on the nonpolar face of the helix and the bulk of the nonpolar face. Studies using large unilamellar phospholipid vesicles showed that the inclusion of cholesterol greatly inhibited leakage by the two Glu-containing peptides. These results cannot be attributed to changes in the phase behavior of the lipids caused by the inclusion of cholesterol or to differences in the secondary structure of the peptides. These results suggest that eukaryotic cells are resistant to lysis by magainins because of peptide-cholesterol interactions in their membranes that inhibit the formation of peptide structures capable of lysis, perhaps by hydrogen bonding between Glu and cholesterol. Bacterial membranes, lacking cholesterol, are susceptible to lysis by magainins.     

Pharmacological aspects of mouse hind-paw oedema induced by Lachesis muta rhombeata venom

Pharmacological aspects of mouse hind-paw oedema induced by subplantar injections of Lachesis muta rhombeata (LMR) venom were investigated. The oedema induced by subplantar injections of 10 to 50 ng/g of LMR venom is dose dependent, with onset, peak and duration at 30, 60 and 180 min, respectively. Subplantar injection of 30 ng/g of Bothrops jararaca (BJ) venom induced oedema that has the same intensity as 30 ng/g of LMR venom but lasts for more than 4 h suggesting different time course. Systemic effects or haemorrhage were not observed with doses less than 50 ng/g. Oedema is not due to the presence of oedematogenic amines since dialysis did not change the oedema induced by 30 ng/g of LMR venom. Part of the oedema induced by LMR venom is due to a thermolabile fraction since pre-heating the venom at 100 degrees C for 15 min induced a significant reduction (56.19 +/- 6.8%) of the oedematogenic activity. The oedema induced by LMR venom is possibly induced by release of a pharmacological active substance at the site of injection. Histamine, arachidonate metabolites, nitric oxide and serotonin may play important roles in the oedematogenic effect of LMR venom since pre-treatment of mice with pyrilamine, indomethacin, dexamethasone, L-NAME and methysergide induced a significant reduction (49.86 +/- 10%; 51.06 +/- 5.9%; 77.66 +/- 3.6%; 73.30 +/- 6.1% and 93.77 +/- 2.8%, respectively) of the oedema formation. The present results demonstrate that the oedema induced by LMR and BJ venoms may be triggered and maintained by different pharmacological mechanisms. Since methysergide and L-NAME were the most active inhibitors of the oedema we can suggest that a link between serotonin release by the venom and a NO synthase activation may be an important step in the oedema formation induced by LMR venom.     

Heavy metals in the hair of opossum from Palo Verde, Costa Rica

Levels of lead, cadmium, mercury, chromium, and manganese were measured in the hair of opossum (Didelphis virginiana) from Palo Verde, Guanacaste, Costa Rica. This area has some agriculture, but is slated for extensive water development that will increase ecosystem exposure to a variety of agricultural chemicals. Metal levels were generally not intercorrelated except for chromium and lead, chromium and cadmium, and cadmium and lead. There were significant gender differences only for lead and chromium, with the significantly smaller females having higher levels. It is suggested that hair from mammals, particularly abundant ominivores may be useful, bioindicators of environmental quality over a long time period.     

Paraquat Exposure of Knapsack Spray Operators on Banana Plantations in Costa Rica

A study of occupational exposure to paraquat was performed among 11 knapsack spray operators at banana plantations in Costa Rica. External and internal exposures were quantified and determinants of exposure identified by measurements, observations, and interviews. Dermal exposure was measured with skin pads, respiratory exposure by personal air sampling, and internal exposure by urine sampling. The wrists, back, and legs were the areas with the highest levels of dermal exposure. Respiratory exposures appeared to be strongly influenced by differences between days, while dermal exposures varied mostly due to differences between plantations. The use of protective clothing did not effectively protect against dermal exposures. Both respiratory and dermal exposures were significantly related to internal exposures, and both should be considered possible routes for systemic absorption of paraquat. It cannot be excluded that measurable levels of exposure can lead to acute as well as chronic health effects. Furthermore, due to poor conditions within the working environment, the spray operators are continuously at risk for high exposures that could lead to severe intoxication, and therefore a strategy for control of exposure is necessary.     

Snakebite mortality in Costa Rica

The mortality rate due to snakebite envenomation in Costa Rica was estimated from 1952 to 1993. The highest mortality was observed during the 1950s and 1960s, with the highest rate of 4.83 per 100,000 population in 1953. In contrast, a rate of 0.2 per 100,000 population per year was estimated from 1990 to 1993. The most conspicuous decline in mortality occurred after 1970. The highest mortality rates were observed in the provinces of Limón and Puntarenas, especially in regions where tropical rain forests had been transformed into agricultural fields. The lowest mortality was in the province of Guanacaste, where tropical dry forest predominates and Bothrops asper (terciopelo), the most important poisonous snake in the country, is not abundant. The majority of fatalities occurred in the age groups from 10 to 19 years old. Males were more affected than females in a ratio of 3.6:1. Before 1980 most fatal cases did not receive medical attention in hospitals, whereas after 1980 the majority of cases with fatal outcome were attended in hospitals.