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  • Open access
  • 27 Reads
Simple analysis system for the rapid and sensitive detection of major ciguatoxin congeners
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Ciguatera is a foodborne poisoning caused by the ingestion of fish contaminated with ciguatoxins (CTXs). Ciguatera is a dire public health issue, since it causes long-lasting neurological effects and more importantly, has no cure. Moreover, ciguatera, earlier considered to be geographically limited in tropical areas, is now being reported outside these locations. This accentuates the need for the development of efficient and rapid methods to detect CTX. Our study is based on a previously established immunoassay for detection of the major CTX congeners (CTX1B and CTX3C series) using magnetic beads and a sandwich format. We have optimized the assay by drastically reducing the time, resources and labour used, establishing a system that can be applied in resource-scarce settings. The 40-minute assay provides a specific and reliable colorimetric signal for CTX1B. The performance of the system has been tested on fish samples and the limit of detection has been found to be 0.01 ppb, which is aligned with the FDA specifications. In addition to this, the reagents have shown appropriate storage stability. Designed to facilitate detection of CTXs, especially in the lower resource settings, our system would proficiently help in the ciguatera surveillance, risk assessment and management in such areas, significantly decreasing health and economic burdens.

  • Open access
  • 19 Reads
Kinetic Characterization of ATPase of Plasmodium falciparum (PfATP4) & its Inhibition by Duttaphrynus melanostictus (Schneider) Skin Extract: An In Vitro Study
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Bufadienolides, which are naturally present in the toxic skin secretions of toads, have recently been discovered to have a variety of antiparasitic effects. These substances are known to inhibit the activity of ATPase enzyme, thereby preventing the ATP usage in the parasites leading to their rapid death. In present study, the crude hydroalcoholic skin extract of Common Asian Toad, Duttaphrynus melanostictus, containing bufadienolides was evaluated for its ability to inhibit the ATPase of Plasmodium falciparum (PfATP4). PfATP4 was isolated from the trophozoites of Plasmodium falciparum 3D7 cells and its kinetic characterization was performed at varying concentrations of ATP, sodium, potassium, hydrogen, and calcium. The results obtained confirmed that PfATP4 followed Michaelis-Menten kinetics when treated with ATP, sodium, and hydrogen while no significant change in the activity was observed after the treatment with potassium and calcium. The inhibition constant of the extract was determined in vitro which was found to be 0.06 µg/ml. The Michaelis-Menten, Lineweaver-Burk and EisenthelCornish-Bowden plots showed that the Km value of the enzyme significantly increased while the Vmax remained unaffected after extract treatment. Therefore, from this preliminary study, it could be concluded that the bufadienolides present in the skin extract possess a potential of being a strong competitive inhibitor of ATPase in Plasmodium falciparum and hence could be further explored as a novel antimalarial drug.

  • Open access
  • 19 Reads
Innovative method of high sensitivity and separation detection for ciguatoxin analogues by LC-MS/MS

Ciguatera Fish Poisoning is the world‘s largest food poisoning. The US FDA recommends an acceptable dose of 0.01 µg CTX1B equivalent/kg. The FDA level of sensitivity detection is possible by using LC/MS/MS equipment of one manufacturer, and a method has been reported to detect [M+Na]+ > [M+Na]+. In this study, we found a highly sensitive method for the detection of ciguatoxins, [M+Na]+ >[M+Na]+ or [M+Li]+ > [M+Li]+, by adding very small amounts of alkali metals such as Na+ or Li+ to the mobile phase. This method suggests that CTXs FDA level detection and high separation chromatography may be able to achieve for LC-MS from any manufacturer.

  • Open access
  • 22 Reads
Synergism between food toxicants acting on sodium channels

Ciguatera fish poisoning (CFP) is one of the most frequent poisonings related to fish consumption. It is caused by the ingestion of fish contaminated with ciguatoxins (CTXs) produced by microalgae of the genus Gambierdiscus and Fukuyoa. The characteristics symptoms of the intoxication include gastrointestinal, cardiovascular and neurological disturbances. CTXs act on voltage-gated sodium channels (VGSC) by their binding to the site 5 of their alpha subunit, shifting the activation voltage towards more negative membrane potentials. This effect is enhanced by the coexistence of CTXs with other VGSC modulators which are also food contaminants as pyrethroid compounds, widely used in agriculture. Nowadays, an increasing presence of ciguatoxins in European Coasts and similarly, the increasing use of phytosanitary products for control of food plagues had raised the concerns regarding human health. In this work, the effects of ciguatoxins and pyrethroids in human sodium channels were investigated. The results presented in this study indicate that both types of compounds have a remarkable synergistic effect in voltage-dependent sodium channels. Exposure of human cells to these compounds elicited a decrease in the maximum peak inward sodium currents and hyperpolarized the activation voltage of sodium channels, effects that were boosted by the simultaneous presence of both toxicants. Since the regulatory levels in food for these components are set separately, this study highlights the need to re-evaluate their limits in feedstock considering that they act on the same cellular target.

  • Open access
  • 22 Reads
Ciguatoxins and methods of detection

Voltage gated sodium channels (VGSC) are the cellular target of ciguatoxins, which cause a food poisoning disease known as ciguatera. Nowadays is a human global disease caused by the consumption of contaminated fish that have accumulated ciguatoxins (CTXs). The algae producers of CTXs were initially found in warmer water but nowadays have spread to European waters. The in vitro methods of detection for ciguatoxins, implemented about 30 years ago, are based in a tumoral mice cell line (Neuro 2A). Since CTXs cause the opening of voltage-dependent sodium channels at hyperpolarizing membrane potentials, producing cell membrane depolarization at rest by mainly increasing sodium influx, the Neuro 2A assay must be reconsidered for the detection of ciguatoxins because this cell line has a low expression of VGSCs. In this work, comparing the effect of CTXs in mice and human neuroblastoma cells and in non-tumoral cells expressing the α6 subunit of sodium channels we conclude that the change in membrane potential is the reliable method to assess the effects of ciguatera toxins by using in vitro techniques employing cells with sodium channels. Therefore the in vitro methods for detection of ciguatoxins must be re-evaluated since it has been demonstrated that the current methodology did not reflects the real CTXs toxicity. Other methods based on membrane depolarization may be more suitable to assess ciguatoxin cell exposure

  • Open access
  • 18 Reads
Insect larvae as Biofactories to produce sphingomyelinase D for Loxosceles antivenom development
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Loxosceles species, commonly named “violin” spiders, are widely spread venomous spiders. Clinical cases of Loxosceles bites are more commonly reported in the Americas, particularly in Argentina, Brazil, Chile and Perú. Most accidents are characterized by dermonecrotic lesions, often referred to as necrotic or gangrenous arachnidism. However, in about 10% of the cases a more serious presentation occurs, characterized by a systemic evolution of the pathology that can result in a fatal outcome, mostly in children and elders. Antivenom against Loxosceles species is being produced in the Americas since the early 1960s. Its Active Pharmaceutical Ingredients are immunoglobulins or their fragments obtained from equine plasma of animals hyperimmunized with spiders’ venom. Venom extraction, a very laborious task with very low yields per spider, is the most relevant production bottleneck. Herein we optimized a biotechnological process based on Spodoptera frugiperda larvae as biofactories to obtain a recombinant version from L. laeta sphingomyelinase D (rSmase). The rSmase was recovered high purity level (94.5%) in one-chromatographic step at low cost. Our version of rSmase was able to induce a neutralizing humoral response in horses’ sera. The amount of venom of 17000 spiders could be replaced with rSmase from 1600 insect larvae. The use of rSmase is expected to change traditional antivenom production.

  • Open access
  • 13 Reads
Production of Micrurus snake venom in Argentina. Preliminary report.
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Micrurus envenoming is a medical emergency that requires the rapid use of antivenom and occasionally the use of pharmacological and mechanical support due de respiratory paralysis that can occur. The antivenom is the only specific treatment and the most important tool to treat the envenomed patients, specially, those treated in peripheral centers where other kind of support apart the antivenom is not available. By this reason the antivenom is essential for the treatment of this type of envenomation. For the antivenom production is necessary to obtain the venom, which is a difficult task due the scarcity of snakes in nature in Argentina and due the short survival of the specimens in captivity, which limits the venom obtaining and consequently the antivenom production. In order to know the historical income of snakes to our serpentarium and the venom production, we analyzed the records of reception of Micrurus specimens and the their venom production in the INPB during the period 1991-2020. Even though this is a preliminary report based in historical registers, initial data provide a draft idea of the coral snakes reception and the Micrurus venom production. In this period was registered the reception of 322 specimens (around 11/year, Md 7, min. 0, max. 76) and in 7 years, no snakes were received. The reception drastically decreased, in the last three decades were received 220 (1991-2000), 89 (2001-2010) and 13 (2011-2020) coral snakes. The number of Micrurus used by milking session varied from 1 to 22 snakes (Md 4), with around 2 milking sessions/year. The individual venom yield varied from 1 to 40 mg (the last in only one individual case). The yield of venom/snake (mg of venom/number of snakes milked) was 8.6 ± 5.5 (Md 8, min 1 max. 26, 25% 4, 75% 11.7). The number of snakes received in the serpentarium decreased from a mean of 34 snakes/year in the period 1991-1995 to 1 snake/year in the period 2016-2020. Despite the difficult in venom obtaining, the Micrurus antivenom was produced with continuity and no deaths by coral snake bites were registered in the last 25 years. The decrease in the number of coral snakes received in the last years may impairs the antivenom production.

  • Open access
  • 8 Reads
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We studied the neutralizing capacity of three anti-Bothropic AVs (antivenoms): 1) BIV, produced by immunizing horses with venoms of Bothrops alternatus [Ba] and B. diporus [Bd) and 2) TTV, produced by immunizing the both mentioned venoms) plus B. jararaca [Bja] and B. jararacussu [Bju] venoms. Their neutralizing capacity on lethality, hemorrhagic, coagulant (plasma), proteolytic and PLA2 activities of Ba, Bd, Bn, Bja and Bju venoms, were studied. In addition the toxicity of Bja and Bju venoms on myolblast C2C12 cells and their neutralization by AVs was assayed. In all the cases BIV and TTV neutralized all the activities of the venoms. Volumetrically the neutralizing doses (mg of V [venom] neutralized by ml of AV [antivenom]), TTV showed more potency. However, when the neutralizing potency was expressed as the mg of AV, the doses turn very close. The ED50s (expressed as mg of AV) for Ba V were= 2.0(1.9-2.2) for BIV and 2.2(2.1-2.3) for TTV. For Bd= 1.9(1.7-2.1) for BIV and 1.4(1.3 to 1.6) for TTV; Bn= 2.7(2.0-2.9 for BIV) and 1.9(1.7-2.1) for TTV; Bja=1.5 (1.5-1.6) for BIV and 1.4(1.4-1.5) for TTV; Bju=3.8(3.4-4.2) for BIV and 3.5(3.4-3.7) for TTV. The hemorrhage and myotoxicity in C2C12 was well equally neutralized (p < 0.05). PLA2 activity (radial hydrolysis of phospholipids and indirect hemolysis) and plasma coagulation were neutralized in all the cases and all the doses got close when were considered as the amount of AV protein required for neutralizations. Bjca and Bju venoms were well neutralized by the BIV, which does not include them in their immunogenic mixture while Bn V was well neutralized by both AVs. The results showed that the cross neutralization on this venoms is high. Differences in neutralization, considering the volume of AV required to neutralize, were strongly related with the amount of specific F(ab´)2 fragments in the AVs.

  • Open access
  • 20 Reads
New Toxicological Approach for the Detection of Tetrodotoxin and their Analogues in PufferFish

Tetrodotoxins (TTXs) are one of the most potent marine neurotoxins known, being responsible for many poisoning accidents and some fatalities. Until now, more than 30 TTX analogues have been described but their individual implication in poisoning is not clear. In order to evaluate the individual toxicity of five TTX analogues purified form the liver of a toxic pufferfish (Lagocephalus sceleratus) caught in Greek waters, a new single-cell biosensing platform based on patch clamp technology has been used. The recording of electrophysiological sodium currents of cells enabled the determination of the Toxicity Equivalency Factor (TEF) of each one of the tested analogues. Additionally, the single-cell biosensing platform proposed in this work was applied to the analysis of different pufferfish tissues, providing composite toxicological responses. Overall, this new biosensing device proved to be useful for toxicological analysis, being promising for their implementation in research and monitoring programs.

  • Open access
  • 22 Reads

Carry-over of Ochratoxin A to Plasma, Serum, Milk, Urine and Faeces in Lactating Cows Fed with Concentrate-rich Diets

Ochratoxin (OT) A is a toxin produced by several species of fungi, including Aspergillus and Penicillium, and can contaminate various food and feedstuffs. Its hepatotoxic, nephrotoxic, and teratogenic effects in animals are well documented. Ruminants possess physiological (i.e., microbial) systems for detoxifying mycotoxins. This experiment investigated the transmission of OTA and its main metabolite OTα into plasma, serum, milk, urine and faeces in lactating Simmental cows. The animals were fed with OTA-containing concentrate-rich diets composed of grass silage, corn silage and concentrate at 37.1%, 12.5% and 50.4% on dry matter basis, respectively. Twelve cows were randomly assigned to two equal groups (OTA-Low/OTA-High), which received 250 and 2500 µg/kg DM diet for seven days, respectively. The concentrations of OTA and OTα in the mentioned biological matrices were measured via high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS/MS). In addition, daily feed intake, chewing behaviour, milk production and clinical parameters were evaluated daily. Milk composition, haematological parameters and hepatic enzymes were evaluated on days 0 and 7 of the experiment. Using three cows per group, the analytical method was validated by determining the following limit of quantification (LOQs) for OTA and OTα: In plasma, serum, and milk 0.5 ng/mL, urine diluted to 10 mM creatinine 5 ppb and in dried faeces 50 ng/g. All stated LOQs are applicable for both analytes. On day 7, the average (± SD) concentrations of OTA in plasma, serum, milk (ng/mL), urine (ppb diluted to 10 mM creatinine) and faeces (ng/g DM) in the group OTA-High were 8.1(±0.9), 7.0(±1.1), 0.0(±0.0), 9.9(±3.2), 222(±94.3), respectively, and for OTα, 18.3(±1.5), 24.8(± 5.0), 4.8(±1.4), 1271(± 609), 1523(±314), respectively. Taken together, ingesting concentrate-rich diets containing up to 2500 μg OTA/kg DM diet for seven days did not affect feed intake, milk production and composition, chewing behaviour, clinical and haematological parameters, or hepatic function of cows. Because most OTA was microbially metabolized, low concentrations were detected in plasma/serum, and carry-over into milk was not detected. The excretion was mainly as OTα in urine and faeces.