Ability of horse anti-Tityus discrepans F(ab')2 ELISA assay to recognize Tityus discrepans venom toxins.

Anti-Tityus discrepans F(ab')2 ELISA recognition of T. discrepans toxins was measured with regression analysis and its slope called ELISA recognition value (ERv). Fractions containing toxins affecting mammal macrophages or Na(+)-channels have Ervs >19. Toxins affecting potassium channels or insect NaV channels have ERvs <10. Fractions including curarizing or antineoplasic peptides had ERvs <1. Erv increases in proportion to mammalian toxin toxicity rather than to toxin molecular mass.

Bactridine's effects on DUM cricket neurons under voltage clamp conditions.

We describe the effects of six bactridines (150 nM) on cricket dorsal unpaired median (DUM) neurons. The addition of bactridine 2 to DUM neurons induced a large current component with a reversal potential more negative than -30 mV, most evident at the end of the pulses. This current was completely suppressed when 1 µM amiloride was applied before adding the bactridines. Since the amiloride sensitive current is able to distort the aim of our study, i.e. the effect of bactridines on sodium channels, all experiments were done in the presence of 1 µM amiloride. Most bactridines induced voltage shifts of V(1/2) of the Boltzmann inactivation voltage dependency curves in the hyperpolarizing direction. Bactridines 1, 4 and 6 reduced Na current peak by 65, 80 and 24% of the control, respectively. The sodium conductance blockage by bactridines was voltage independent at potentials >20 mV. Bactridines effect on cricket DUM neurons does not correspond to neither α- nor ß-toxins. Most bactridines shifted the inactivation curves in the hyperpolarizing direction without any effects on the activation m(∞)-like curves. Also bactridines differ from other NaScpTx in that they increased an amiloride-sensitive conductance in DUM neurons. Our result suggest that the α/ß classification of sodium scorpion toxins is not all encompassing. The present work shows that bactridines target more than one site: insect voltage dependent Na channels and an amiloride-sensitive ionic pathway which is under study.

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.

Antigenic cross-reactivity between sixteen venoms from scorpions belonging to six genera.

Venoms of 15 scorpion species from Venezuela and one from Brazil were compared in their antigenic cross-reactivity with specific F(ab')2 against Tityus discrepans (Td-antibodies), using the method of King and collaborators (1). Our results show that Tityus venoms cross-reactivity (shared epitopes) with the venoms of other species within the genus tended to be less for a greater distance between the habitat of the species. A nonparametric linear regression of free Td-antibody binding to T. discrepans venom immobilized to a solid phase in the presence of other Tityus venoms versus distance showed binding = a + b x log10 (distance) where: median (95% confidence interval) for a = 0.92 (7.43, 9.80) and b = 17.20 (4.15, 22.57) binding/log10(Km); Spearman rS = 0.783 with associated P = 0.006. Our results show that toxins from different Tityus species, targeting mammalian Na+ and K+ channels, are antigenically very similar. Venoms from species from other genera such as Centruroides, Broteas, Diplocentrus, Chactas, and Rhopalurus did not cross-react with Td-antibodies.

Purification of a myotoxin from the toadfish Thalassophryne maculosa (Günter) venom.

Venom was milked by gently pressing the base of the opercular and dorsal fin spines. Three fractions were obtained by molecular exclusion high pressure liquid chromatography (HPLC) (Protein Pak 125SW, Millipore Corporation) column, but only the last one with 22.7 min retention time (rt) was biological active (TmPP-22.7). This fraction was rechromatographed on reversed phase HPLC chlorobutylsilane columns (C4, Vydac) nine fractions were obtained, but only one (TmC4-47.2) with 47.2 min rt was biologically active. MALD-TOF mass analysis was carried out on two samples of TmC-47.2 and the results were 15,161.36 and 15,154.70 a.m.u., respectively. Raw venom (1040 microg/ml) depolarised frog (Hyla crepitans) muscle irreversibly from -85 (-88, -81) mV (n=20, median and its 95% CI) to -18 (-24, -15) mV (n=24). The biological activity in TmPP-22.7 (38 microg/ml), which depolarised muscle fibres from -79 (-82, -76) mV (n=20) to -63 (-69 -57) mV (n=24). The depolarising fraction was TmC4-47.2 (50 microg/ml) which depolarised muscles from -87 (91, -82) mV (n=33) to -63 (-76 -51) mV (n=53); the depolarising effect at this concentration was completely reversed on washing with normal saline for 2 h. Muscles treated with 1 microM tetrodotoxin (TTX) were depolarised from -80 (-85, -72) mV (n=49) to -44 (-56, -31) mV (n=44) when 100 microg/ml TmC4-47.2 were applied with TTX; washing 130 min with 1 microM TTX repolarised to -59 (-69, -50) mV (n=25). We also present evidence that TmC4-47.2 induces myonecrosis in mice.

Histopathological changes and inflammatory response induced by Tityus discrepans scorpion venom in rams.

Anesthetized rams envenomed s.c. with 40 microg/kg Tityus discrepans scorpion venom developed fasciculation, hypothermia, polyuria, pulmonary wet rales, tachypnea, respiratory distress and arrhythmia. Rams developed a cascade of inflammation reactions, characterized by activation of macrophages, fibroblasts and neutrophils, neutrophil infiltration and aggregation, vasculitis, arteritis and abundant fibrin deposition. At the inoculation site, venom was detected by immunohistochemistry in the extra cellular matrix, lymphatic vessels' and venules' lumen, inside macrophages and surrounding nerves. Extra cellular matrix was degraded at the inoculation site perhaps by activated neutrophils. Envenoming produced hepatocytes with Mallory body-like vacuoles which may be due to the increased plasmatic levels of TNF-alpha and IL6. Venom produced degranulation and vacuolization of acinary cells as well as interstitial swelling and necrosis. Necrosis of the Langerhan's islets occurred occasionally. Lungs showed the most deleterious effects developing wall collapse and necrosis, diffuse injury of the alveolar capillary barrier, interstitial and alveolar fibrin deposits with strong neutrophil infiltration. Massive infiltration of lymphocytes and macrophage occurred in the intestinal submucose, to the point that it modified villi and intestinal folding morphology. Envenomation developed a marked leukocyte aggregation surrounding nerves at the inoculation site. This study reveals that beyond its neurotoxicity, Tityus venom produces a severe and widespread inflammatory syndrome, expressed as histopathological changes at the site of inoculation, as well as in remote organs such as pancreas, lungs, intestine and liver. Our results suggest that not all remote targets are directly affected by the venom but that, as proposed earlier, are modified by inflammation by products produced elsewhere.

Modelling Tityus scorpion venom and antivenom pharmacokinetics. Evidence of active immunoglobulin G's F(ab')2 extrusion mechanism from blood to tissues.

Modelling Tityus scorpion venom and antivenom pharmacokinetics. Evidence of active immunoglobulin G's F(ab')(2) extrusion mechanism from blood to tissues. We measured pharmacokinetic parameters for T. discrepans venom in rams. Forty, 75 or 100 microg/kg venom were injected subcutaneously in the inner side of the thigh. Plasma venom content (venenemia) was determined by enzyme-linked immunosorbent assay (ELISA) from 0 to 300 min after injecting venom. Venenemia was fit to a three-compartment model (inoculation site, plasma and extra vascular extracellular space), it was assumed that the venom may also be irreversibly removed from plasma. Calculated time course of venom content shows that at any time no more that 30% of the venom is present in plasma. Venenemia peaks at 1h and decays afterwards. Fluorescently labelled antivenom [horse anti-TityusF(ab')(2) or fraction antigen binding, immuglobulin without Fc chain covalently bound to fluorescine or fluorescamine] pharmacokinetics was determined. Although F(ab')(2) molecular weight is >/=10 times bigger that toxin's, the rate of outflow of F(ab')(2) from blood to tissues was approximately 4 times faster than the venom's outflow. Venom content in the injection site decays exponentially for >6h, this prediction was confirmed immunohistochemically. Only approximately 5% of the venom is eliminated in 10h; approximately 80% of the venom is in the tissues after 2h and remains there for >10h.

Ardiscretin a novel arthropod-selective toxin from Tityus discrepans scorpion venom.

A new arthropod selective toxin was purified from the venom of the Venezuelan scorpion Tityus discrepans, and its amino acid sequence, cDNA clone and biological activity are reported here. The amino acid sequence of this peptide, named ardiscretin (from arthropod toxin of T. discrepans) was completed by Edman degradation and mass spectrometry. It is a single polypeptide composed by 61 amino acids with an amidated cysteine residue at the C-terminal end, closely packed by four disulfide bridges. The atomic mass unit (a.m.u.) experimentally determined was 7103.8 a.m.u. This peptide was shown to be specific for invertebrates (crickets, triatomides, crabs and squids), but non-toxic to mice, at the dose assayed. Ardiscretin inhibits the Na(+)-currents of squid giant axons in an apparent irreversible manner, whose inhibitory effect is reached at 30 microM toxin concentration. Sequence comparison showed that it is phylogenetically closely related to insect-specific scorpion toxins. Ardiscretin produced a small depolarization and induced repetitive firing in squid axons resembling those of DDT [1,1'(p-chlorobenzyl)2-tricloretane] in its ability to slow down action potential, to induce repetitive firing, and in that the concentration required for any effect in squid axon is rather high.

Identification of Enterobacter bacteria as saxitoxin producers in cattle's rumen and surface water from Venezuelan Savannahs.

We have previously shown that a paralytic toxin able to block sodium channels in nerve is associated with a cattle disease known as bovine paraplegic syndrome (BPS) [Toxicon. 31 (1993) 1581]. We have now identified this as saxitoxin (STX) using HPLC by either the methods of [Toxicon. 31 (1993) 1581], or [Toxicon. 25 (1987) 1105]. In recent experiments we were able to collect and cultivate facultative anaerobic bacteria growing on rumen, grass and ponds of corrals with high incidence of BPS; the cultured bacteria produce compounds indistinguishable from STX under both HPLC procedures described above. Two species of the Enterobacter genus (E. asburiae and E. cloacae) and a strain of Klebsiella pneumoniae, were identified using standard biochemical criteria as well as gas chromatography of bacterial lipids. All these bacteria produced STX in aerobic cultures.

Relationship between plasmatic levels of various cytokines, tumour necrosis factor, enzymes, glucose and venom concentration following Tityus scorpion sting.

A sandwich enzyme-linked immunosorbent assay was developed for measuring Tityus venom levels in plasma. The method proved capable of distinguishing patients with only local symptoms from controls, and was used to quantify venom in 205 accidental human envenomations. Our results show that the severity of envenoming is related to the patient plasma venom concentration. This depends on time elapsed between the sting and when the plasma was drawn. We observed that 46 and 49% of patients with moderate to severe symptoms (MS, n=41) showed hyperamylasemia and hyperglycemia, respectively. In addition, 39% of cases with MS symptoms had partial thromboplastin time values prolonged or shorted and 6.5% of patients with local symptoms (LS, n=164) had only prolonged prothrombin time values. Interleukin 6 (IL6) increased significantly in patients with MS symptoms. IL6 values increased with hyperamylasemia, envenoming severity and time hyperamylasemia.

Los pacientes de escorpionismo con sintomatología local tiene niveles importantes de veneno en plasma / The scorpionism patients with local symptomatology have important venoms levels in plasma

Se desarrolló un método de inmunoensayo (Enzime-Linked Imunosorbent Assay) sensible y específico para cuantificar veneno de T. discrepans en plasma de pacientes de escorpionismo. Los casos se clasificaron como: asintomáticos (Clasificación del Hospital "Dr. Leopoldo Manrique Terrero", Caracas, D.F.) equivalente a sintomatología local (Clasificación del Hospital "Victorino Santaella", Los Teques, Edo. Miranda), o con sintomatología sistemática: leve, moderada o grave. Se analizó el plasma de 82 pacientes, de los cuales 23 fueron niños. La venenemia en 58 pacientes con sintomatología local estuvo entre 0,01 y 17,2 ng/ml con una mediana e intervalos de confianza de 95 por ciento (entre paréntesis) de 0,5 (0,2 - 2)ng/ml. La venenemia en 13 pacientes con sintomatología leve o moderada flactuó desde 0,1 hasta 202 ng/ml [11,2 (0,5 - 80,4) ng/ml]. Sólo se obtuvo plasma de un paciente con sintomatología grave el cual presentó una venenemia de 31,8 ng/ml. El nivel de falsos positivos del ensayo se determinó con el plasma de 10 personas no emponzoñadas en las cuales el ensayo indicó una venenemia aparente de 0,13 (0,5 - 0,25) ng/ml. Las concentraciones más altas y después de aplicar antiveneno (n= 7) demostró que éste hace desaparecer el veneno del plasma. Se observaron alteraciones en el tiempo parcial de tromboplastina prevalentemente en pacientes con sintomatología local. Alteraciones en amilasa y glicemia sólo se observaron en pacientes con clínica sistémica. El 14 por ciento de 58 pacientes con sintomatología local presentaron alteraciones del tiempo parcial de tromboplastina indicativas de anticoagulación en algunos, o procoagulación en otros casos; estos coincide con resultados in vitro de nuestro laboratorio donde se identificaron fracciones pro y anticoagulantes en el veneno de Tityus discrepans (D'Suze y col., 2000). En 63 por ciento de los pacientes con sintomatología local se detectó veneno en el plasma, 24 por ciento de ellos en concentraciones entre 1 y 4 ng/ml y 9 por ciento por encima de ese rango, estas concentraciones de veneno se correlacionan en otros estudios con aumentos de citokinas, factores de necrosis tumoral y de agregación plaquetaria. Este trabajo indica que la ausencia de clínica no es equivalente a ausencia de veneno en plasma ni a ausencia de daño. Este trabajo fue financiado por el proyecto aplicado IVIC-Silanes

Venezuelan arachnids. Two new species of the Tityus Genus (Scorpionida: Buthidae) and the chromatographic profile of venom as a possible taxonomical tool

Two new species of the Tityus genus are described. T. isabelceciliae n. sp lives on the northern central slope of the Cordillera de la Costa. It belongs to the discrepans group and is dangerous to man due to its high number, aggressive behavior, domiciliary habits, and high toxicity of its venom. T. isabelceciliae venom is similar to other Tityus in relation to the molecular weight range and the biological activity of its components. However, the proportions of each fraction in the venom pooled from many T. isabelceciliae differ from the proportions in other Tityus, indicating that these proportions may have a taxonomical value. The venom LD50 is 38.1 (36.3, 39.9) µg/g mouse (Death in 30 min, Dixon and Mood (14) sequential method, median and 95 per cent confidence interval, n=7). Venom production was 916 (625, 1213) µg protein per animal (n=38): females [944 (750, 1150) mg protein per animal, n=24] and males [824 (550, 112) mg protein per animal, n=14] did not differ in venom production (P>0.05). There was no correlation between animal total weight and venom production. T. rusmelyae n. sp. from the androcottoides group lives near the town of Humocaro Alto in the Lara State, Venezuela. The male specimens have clearly defined keels and granules. It differs from other species of this genus in that the prominent characteristics are observed in male specimens.

A novel K+ channel blocking toxin from Tityus discrepans scorpion venom.

A novel toxin (TdK1) was purified from the venom of the scorpion Tityus discrepans, sequenced and functionally characterized. It contains 37 amino acid residues and blocks reversible the shakerB K+ channel expressed in SF9 cells with a Kd in the order of 280 nM. The proposed systematic nomenclature for this peptide is alpha-KTx4.3.

Tityus discrepans venom produces a respiratory distress syndrome in rabbits through an indirect mechanism.

It is well known that scorpion venom induces lung lesions and respiratory distress which are usually classified as pulmonary oedema (PO). Tityus discrepans is a scorpion that lives in the north-central area of Venezuela, is the most common source of human envenomation here and produces PO. We studied the action of the venom of Tityus discrepans on whole rabbits and on their isolated lungs perfused with Krebs saline with 1 g/l of bovine serum albumin (Krebs-BSA saline). Two milligram of venom were diluted in 250 ml of solution (approximately the rabbit's total blood volume) and used to perfuse isolated lungs. Lung oedema occurred in rabbits which received 1 mg/kg of scorpion venom i.p., heparin prevented the production of this lung oedema. T. discrepans venom produced PO, in rabbits pretreated with 15 mg/kg of ajoene. Yet, Tityus venom had no effects on isolated lungs perfused with citrated or heparinized blood, and in lungs perfused with Krebs-BSA with normal Ca2+. These result show that Tityus venom does not act directly on lungs. Otherwise, we have observed that abundant microthrombi occurred in all rabbit lungs exposed to venom in vivo, suggesting that these clotting alterations are fundamental to produce PO. The presence of intravascular microthrombi is not characteristic of the usual PO hinting that scorpion venom induced pulmonary alterations are a different clinical entity. We thus propose that the use of the term pulmonary oedema in scorpionism should abandoned in favor of scorpion venom respiratory distress syndrome.

Annual variations of the sodium channel blocking toxin in Venezuelan cattle prone to develop the bovine paraplegic syndrome: its dependence on rain

A characteristic of the bovine paraplegic syndrome (BPS) is ventral or sternal decubitus in animals that make unsuccessful efforts to stand when stimulated. Death occurs after a few days. In a previous work we showed the existence of a sodium channel blocking toxin (SCBT) produced by ruminal bacteria in cattle with or liable to develop BPS. The presence of SCBT in bovine plasma sampled monthly during the rainy and dry seasons in animals without BPS has now been observed. A positive correlation between rain precipitation and plasma toxin levels (Spearman's rank correlation coefficient=0.457, n=135, p<10(-5) was found. Precipitation was 194 (121-289) mm water/month (n= 162 months in 27 years, median and its 95 per cent confidence interval) during the rainy season and 7 (0-35) mm water/month (n=109 months in 28 years) during the dry season [p < 10(-6), Mann-Whitney (Wilcoxon) test]. Plasma toxin levels were determined by high performance liquid chromatography (HPLC). Toxin levels, expressed as peak units of area (PUA) at 340 nm, (1 PUA = 2.5 x 10(-6) units of absorbance/cm) were 5.5 (0.0 - 23.3) PUA/nl (n = 133) in the rainy season and 0.0 (0.0 - 1.1) PUA/nl in the dry season (n=88, p=2x10(-6)). The distribution of toxin concentration in both groups was also different (X² test, p=0.024,7, 7 degrees of freedon). It is then determined that toxin was not detectable in 84.1 per cent of the cows in the dry season (n = 88) and in only 38.3 per cent of the cows in the rainy season (n = 133) (X² test, p < 10(-6)).

Isolation and characterization of a potent curarizing polypeptide from Tityus discrepans scorpion venom.

The pure TdI-1 polypeptide that blocks miniature endplate potentials (MEPPs) and abolishes or reduces endplate potentials (EPPs) below the action potential threshold was identified from the crude fraction of Tityus discrepans venom. The toxin is a potent reversible non-depolarizing muscle relaxant that blocks more than 95% of the EPP at a 2 microM (0.1 mg/ml) concentration. On a molar basis, TdI-1 is as potent as or more potent than many muscle relaxants since, at the concentration used, the toxin suppressed more than 95% of the EPP. Using matrix-assisted laser desorption time of flight (MALD-TOF) ionization mass spectrometry, TdI-1 was found to have an unusally large mol. wt for a scorpion toxin, close to 48,000. The N-terminal sequence of the first 23 residues of TdI-1 was also determined. The fragment differs from the N-terminal sequences of all 140 peptidic scorpion toxins found in the SWISSPROT and PIR databases using the search engine of the felix.EMBL-Heidelberg.de computer (European Molecular Biology Laboratory, Heidelberg, Germany.

High performance liquid chromatography purification and amino acid sequence of toxins from the muscarinic fraction of Tityus discrepans scorpion venom.

Tityus discrepans venom was fractionated by gel filtration on Sephadex G-50 column. The peptides in fraction II from Sephadex were further purified by high performance liquid chromatography, through a C4 reverse-phase column. Lethality of purified peptides was determined by injection into mice and crabs, and their effects were verified electrophysiologically on frog (Hyla crepitans) sartorius neuromuscular junction. Toxins having retention times between 39.6 and 40.7 min depolarized the muscle membrane and caused acetylcholine release at the endplate. The toxin eluted at 42.67 min increased the frequency of miniature endplate potentials without depolarizing muscle fibres. The four most active toxins were reduced, carboxymethylated and sequenced by automatic Edman degradation and named TdII-1 to II-4. Toxin gamma from Tityus serrulatus venom and the toxins from T. discrepans venom were found to be structurally distinct. TdII-1 to II-4 lack the pancreatic effects of T. serrulatus' toxin gamma; yet, the five toxins act on Na+ channels.

Presence of curarizing polypeptides and a pancreatitis-inducing fraction without muscarinic effects in the venom of the Venezuelan scorpion Tityus discrepans (Karsch).

Four toxic polypeptidic fractions (TdF-I-IV) were purified from the venom of the Venezuelan scorpion Tityus discrepans by means of gel filtration on Sephadex G'-50. The peptides have mol. wts of approx. 6000 and normalized elution volumes (Vn = Elution volume/Total column volume) of: TdF-I = 0.27 (0.26, 0.28), n = 13; TdF-II = 0.40 (0.39, 0.41), n = 15; TdF-III = 0.57 (0.56, 0.59), n = 14, and TdF-IV = 0.68 (0.67, 0.70), n = 13 (median and its 95% confidence interval, n = number of elutions used to calculate the median). Mice (white, male, 16-19 g, IVIC strain) were injected with these fractions and sacrificed 48 hr later. No toxicity was observed when fraction I (0.93 microgram/g mice) or IV (2.51 micrograms/g mice) was injected i.p. into mice. TdF-II (9 to 50 micrograms/g mice) produced sialorrhea, dyspnea and death 1 hr after i.p. injection. Light microscopy of the pancreas revealed that TdF-III (3.42 micrograms/g mice) produced structural modifications such as acinar cell vacuolization, degranulation and interstitial swelling; these changes are characteristic of acute pancreatitis. No effects on the islets of Langerhans or the pancreatic ducts were observed. TdF-III had no overt muscarinic effects when injected i.p. into mice. On the neuromuscular preparation of the frog (Hyla crepitans) TdF-I blocked neuromuscular transmission at the postsynaptic membrane; TdF-II depolarized the muscle membrane by opening sodium channels and TdF-IV prolonged action potentials, suggesting potassium channel blockage.

Specific blockage of squid axon resting potassium permeability by Haliclona viridis (Porifera: Haliclonidae) toxin (HvTX).

The action of partially purified HvTX, toxin of the marine sponge H. viridis, was explored on the giant axon of the tropical squids Doryteuthis plei and Sepioteuthis sepioidea. HvTX depolarizes the nerves dose dependently. The effect occurs after blocking sodium channels with tetrodoxin (1 microM), removing external Na+, blocking electrically excitable K+ channels with 3,4-diaminopyridine (10 mM) or internal and external application of tetraethylammonium (40 mM). Ouabain (up to 10 mM) does not modify HvTX effect. The action of HvTX occurs only when it is applied to the outer phase of the nerve membrane; microinjection of the toxin into the axons lacks depolarizing effects. HvTX reduces the dependence of membrane potential on external potassium concentration. The apparent 86Rb+ permeability (pi') was measured in axons of S. sepioidea. The value of pi' in normal artificial sea water was 80 (61,96) nm/sec (median and its 95% confidence interval, n = 8) and raised to 1030 (588, 2113) nm/sec (n = 7) when the axons were depolarized to 0 mV raising external K+ to 300 mM. In axons depolarized with HvTX (10 mM external K+) to 0 mV, pi' was 88 (55, 97) nm/sec (n = 8). HvTX could not prevent (P >> 0.05) the increase in pi' induced by 300 mM K+ when the ion concentration was raised before toxin application [pi' = 660 (354, 1876) nm/sec, n = 7]. Most of the 86Rb+ permeability increase in high K+ was prevented if HvTX was added before external K+ was raised [pi' = 298 (264, 337) nm/sec, n = 8]. All the measures of pi' were carried out in solutions containing 1 microM tetrodotoxin, 1 mM 3,4-diaminopyridine and 2 mM ouabain.

Toxicology of a bovine paraplegic syndrome.

A clinical entity named 'bovine paraplegic syndrome' ('síndrome parapléjico de los bovinos') has spread alarmingly in the cattle-growing areas of the central and eastern plains of Venezuela. It is estimated that four million cattle are bred in the area where the disease occurs. The mortality ranges from 5 to 25% of the animals at risk, mostly pregnant or lactating cows. The principal characteristic of the bovine paraplegic syndrome is ventral or sternal decubitus, in animals that make vain efforts to stand when stimulated. The diagnosis is established when all other possible causes (e.g. paralytic rabies, botulism and blood parasites such as Anaplasma marginal, Babesia bovis, B. bigemina, and Trypanosoma vivax) have been ruled out clinically and by laboratory tests. Death always occurs, usually after a few days, and there is no known treatment. In this work, we describe results that show the presence of a toxin in the cattle suffering from, or liable to suffer from the syndrome. The toxin is produced by ruminal bacteria. In squid giant axons under voltage clamp conditions, the toxin blocks the sodium current. We detected the toxin analytically by absorbance measurements at 340 nm after reacting with picrylsulfonic acid. We obtained a good separation of the toxin with isocratic high pressure liquid chromatography, using 40% methanol in water on phenylborasil columns.