Toxins

782 KiB

Open AccessArticle

Computational Design of Peptide Ligands for Ochratoxin A

by Meike Heurich, Zeynep Altintas and Ibtisam E. Tothill

Toxins 2013, 5(6), 1202-1218; https://doi.org/10.3390/toxins5061202 - 21 Jun 2013

Cited by 38 | Viewed by 9172

In this paper, we describe a peptide library designed by computational modelling and the selection of two peptide sequences showing affinity towards the mycotoxin, ochratoxin A (OTA). A virtual library of 20 natural amino acids was used as building blocks to design a [...] Read more.

In this paper, we describe a peptide library designed by computational modelling and the selection of two peptide sequences showing affinity towards the mycotoxin, ochratoxin A (OTA). A virtual library of 20 natural amino acids was used as building blocks to design a short peptide library against ochratoxin A template using the de novo design program, LeapFrog, and the dynamic modelling software, FlexiDock. Peptide sequences were ranked according to calculated binding scores in their capacity to bind to ochratoxin A. Two high scoring peptides with the sequences N'-Cys-Ser-Ile-Val-Glu-Asp-Gly-Lys-C' (octapeptide) and N'-Gly-Pro-Ala-Gly-Ile-Asp-Gly-Pro-Ala-Gly-Ile-Arg-Cys-C' (13-mer) were selected for synthesis from the resulting database. These synthesized peptides were characterized using a microtitre plate-based binding assay and a surface plasmon resonance biosensor (Biacore 3000). The binding assay confirmed that both de novo designed peptides did bind to ochratoxin A in vitro. SPR analysis confirmed that the peptides bind to ochratoxin A, with calculated K_D values of ~15.7 ?M (13-mer) and ~11.8 ?M (octamer). The affinity of the peptides corresponds well with the molecular modelling results, as the 13-mer peptide affinity is about 1.3-times weaker than the octapeptide; this is in accordance with the binding energy values modelled by FlexiDock. This work illustrates the potential of using computational modelling to design a peptide sequence that exhibits in vitro binding affinity for a small molecular weight toxin. Full article

(This article belongs to the Special Issue Recent Advances in Ochratoxins Research)

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(A) Minimized and annealed structure of the ochratoxin A template (shown as stick and ball); (B) The generation of new peptide compounds begins with a pool of potential monomers (amino acids) and a virtual cavity (shown as a box) in which to place them with the target molecule, i.e., ochratoxin A. This virtual screening process is called “electrostatic screening”. The ochratoxin A template is shown in purple interacting with random amino acid monomers (stick and ball) was screened using the LeapFrog design tool. Full article ">Figure 2
Final result of de novo designed peptide sequences shown interacting with ochratoxin A. The [N'-Gly-Pro-Ala-Gly-Ile-Asp-Gly-Pro-Ala-Gly-Ile-Arg-Cys-C'] peptide (A, left) and the [N'-Cys-Ser-Ile-Val-Glu-Asp-Gly-Lys-C'] peptide (B, right) sequence are seen as space-filled, ochratoxin A as stick and ball structures. Ochratoxin A mainly interacts with the peptide backbone of the N-terminal end of the 13-peptide (B) and the central region of the Octamer peptide backbone. Full article ">Figure 3
Capacity of the ochratoxin A-HRP to bind to the octapeptide and 13-mer peptide. The interaction between serial dilutions of HRP-conjugated ochratoxin A and octapeptide (A) and 13-mer peptide (B) immobilized to functionalized microtitre plates via amine coupling (●) and thiol coupling (■) is expressed as absorbance at A 450 nm versus ochratoxin A-HRP concentration (µM). Error bars illustrate the mean and standard deviation of multiple experiments. Full article ">Figure 4
Sensorgrams displaying binding curves of 100 mg L−1 (0.15 μM) ochratoxin A-BSA (reference subtracted) to immobilised peptides, (a) 13-mer peptide GPAGIDGPAGIRC (blue) and (b) octapeptide CSIVEDGL (red); and 100 mg L−1 BSA alone (negative control) binding to (c) 13-mer peptide (dark grey) and (d) octapeptide (light grey). Full article ">Figure 5
Sensorgrams showing the binding interaction of immobilized (A) octamer peptide CSIVEDGL and (B) 13-mer peptide GPAGIDGPAGIRC with decreasing ochratoxin A-BSA analyte concentration (from top to bottom: 100, 1, 0.1, 0.01 mg L−1; equivalent to 15pM–0.15 μM. BSA reference binding is shown in grey. Full article ">

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Open AccessReview

Heparin-Binding Epidermal Growth Factor-like Growth Factor/Diphtheria Toxin Receptor in Normal and Neoplastic Hematopoiesis

by Fabrizio Vinante and Antonella Rigo

Toxins 2013, 5(6), 1180-1201; https://doi.org/10.3390/toxins5061180 - 18 Jun 2013

Cited by 35 | Viewed by 11712

Abstract

Heparin-binding EGF-like growth factor (HB-EGF) belongs to the EGF family of growth factors. It is biologically active either as a molecule anchored to the membrane or as a soluble form released by proteolytic cleavage of the extracellular domain. HB-EGF is involved in relevant [...] Read more.

Heparin-binding EGF-like growth factor (HB-EGF) belongs to the EGF family of growth factors. It is biologically active either as a molecule anchored to the membrane or as a soluble form released by proteolytic cleavage of the extracellular domain. HB-EGF is involved in relevant physiological and pathological processes spanning from proliferation and apoptosis to morphogenesis. We outline here the main activities of HB-EGF in connection with normal or neoplastic differentiative or proliferative events taking place primitively in the hematopoietic microenvironment. Full article

(This article belongs to the Special Issue Diphtheria Toxin)

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647 KiB

Open AccessArticle

Determinants of pH-Dependent Modulation of Translocation in Dermonecrotic G-Protein-Deamidating Toxins

by Tana L. Repella, Mengfei Ho and Brenda A. Wilson

Toxins 2013, 5(6), 1167-1179; https://doi.org/10.3390/toxins5061167 - 14 Jun 2013

Cited by 7 | Viewed by 6927

Abstract

Cytotoxic necrotizing factors from E. coli (CNF1, CNF2) and Yersinia (CNFy) share N-terminal sequence similarity with Pasteurella multocida toxin (PMT). This common N-terminal region harbors the receptor-binding and translocation domains that mediate uptake and delivery of the C-terminal catalytic cargo [...] Read more.

Cytotoxic necrotizing factors from E. coli (CNF1, CNF2) and Yersinia (CNFy) share N-terminal sequence similarity with Pasteurella multocida toxin (PMT). This common N-terminal region harbors the receptor-binding and translocation domains that mediate uptake and delivery of the C-terminal catalytic cargo domains into the host cytosol. Subtle variations in the N-terminal ~500 amino acids of CNFs and PMT could allow for selective recognition of cellular receptors and thus, selective target cell specificity. Through studies with cellular inhibitors, we have identified an additional novel function for this region in modulating responses of these toxin proteins to changes in pH during intoxication and delivery of the catalytic cargo domain into the cytosol. Full article

(This article belongs to the Special Issue Novel Properties of Well-Characterized Toxins)

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Open AccessReview

Staphylococcus aureus ?-Toxin: Nearly a Century of Intrigue

by Bryan J. Berube and Juliane Bubeck Wardenburg

Toxins 2013, 5(6), 1140-1166; https://doi.org/10.3390/toxins5061140 - 13 Jun 2013

Cited by 503 | Viewed by 28117

Abstract

Staphylococcus aureus secretes a number of host-injurious toxins, among the most prominent of which is the small ?-barrel pore-forming toxin ?-hemolysin. Initially named based on its properties as a red blood cell lytic toxin, early studies suggested a far greater complexity of ?-hemolysin [...] Read more.

Staphylococcus aureus secretes a number of host-injurious toxins, among the most prominent of which is the small ?-barrel pore-forming toxin ?-hemolysin. Initially named based on its properties as a red blood cell lytic toxin, early studies suggested a far greater complexity of ?-hemolysin action as nucleated cells also exhibited distinct responses to intoxication. The hemolysin, most aptly referred to as ?-toxin based on its broad range of cellular specificity, has long been recognized as an important cause of injury in the context of both skin necrosis and lethal infection. The recent identification of ADAM10 as a cellular receptor for ?-toxin has provided keen insight on the biology of toxin action during disease pathogenesis, demonstrating the molecular mechanisms by which the toxin causes tissue barrier disruption at host interfaces lined by epithelial or endothelial cells. This review highlights both the historical studies that laid the groundwork for nearly a century of research on ?-toxin and key findings on the structural and functional biology of the toxin, in addition to discussing emerging observations that have significantly expanded our understanding of this toxin in S. aureus disease. The identification of ADAM10 as a proteinaceous receptor for the toxin not only provides a greater appreciation of truths uncovered by many historic studies, but now affords the opportunity to more extensively probe and understand the role of ?-toxin in modulation of the complex interaction of S. aureus with its human host. Full article

(This article belongs to the Special Issue Pore-Forming Toxins)

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855 KiB

Open AccessReview

The Pore-Forming Haemolysins of Bacillus Cereus: A Review

by Nalini Ramarao and Vincent Sanchis

Toxins 2013, 5(6), 1119-1139; https://doi.org/10.3390/toxins5061119 - 7 Jun 2013

Cited by 137 | Viewed by 21510

Abstract

The Bacillus cereus sensu lato group contains diverse Gram-positive spore-forming bacteria that can cause gastrointestinal diseases and severe eye infections in humans. They have also been incriminated in a multitude of other severe, and frequently fatal, clinical infections, such as osteomyelitis, septicaemia, pneumonia, [...] Read more.

The Bacillus cereus sensu lato group contains diverse Gram-positive spore-forming bacteria that can cause gastrointestinal diseases and severe eye infections in humans. They have also been incriminated in a multitude of other severe, and frequently fatal, clinical infections, such as osteomyelitis, septicaemia, pneumonia, liver abscess and meningitis, particularly in immuno-compromised patients and preterm neonates. The pathogenic properties of this organism are mediated by the synergistic effects of a number of virulence products that promote intestinal cell destruction and/or resistance to the host immune system. This review focuses on the pore-forming haemolysins produced by B. cereus: haemolysin I (cereolysin O), haemolysin II, haemolysin III and haemolysin IV (CytK). Haemolysin I belongs to the cholesterol-dependent cytolysin (CDC) family whose best known members are listeriolysin O and perfringolysin O, produced by L. monocytogenes and C. perfringens respectively. HlyII and CytK are oligomeric ß-barrel pore-forming toxins related to the ?-toxin of S. aureus or the ß-toxin of C. perfringens. The structure of haemolysin III, the least characterized haemolytic toxin from the B. cereus, group has not yet been determined. Full article

(This article belongs to the Special Issue Pore-Forming Toxins)

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Haemolytic types (HT) for B. cereus and B. thuringiensis strains isolated from small soil samples. Haemolytic activity was determined at 30 °C on sheep blood agar plates. All 198 strains were plated on blood agar at the same time and compared after 15 h. The haemolytic activity of each strain was estimated twice, and each replicate was classified “blind” with respect to the previous one. Nine different haemolytic types (A to I) were identified among the 198 strains [<a href="#B31-toxins-05-01119" class="html-bibr">31</a>]. Full article ">Figure 2
Structure of the Perfringolysin O molecule shown in ribbon representation. Reproduced with permission from [<a href="#B40-toxins-05-01119" class="html-bibr">40</a>]. Full article ">Figure 3
Schematic representation of the transcriptional regulator PlcR and its cognate cell-cell signalling peptide PapR. The activity of PlcR depends on PapR, a secreted signalling peptide re-imported into the bacterial cell through the Opp system [<a href="#B51-toxins-05-01119" class="html-bibr">51</a>]. After its export, PapR is cleaved to a C-terminal heptapeptide active fragment that accumulates in the medium [<a href="#B52-toxins-05-01119" class="html-bibr">52</a>]. When high bacterial densities are reached, PapR concentration increases inside the bacterial cells, promoting its interaction with PlcR. The PapR-PlcR complex then binds to its DNA recognition site, the palindromic PlcR box, triggering a positive feedback loop that upregulates the expression of a regulon of 45 genes encoding proteins that are essentially secreted or bound or attached to cell wall structures at the interface between the bacterial cell and its environment [<a href="#B55-toxins-05-01119" class="html-bibr">55</a>]. These proteins are likely to be involved in host tissue degradation or in protecting the bacterial cell from host immune defenses, and may act together to provide food supply. Ultimately, when the bacteria enter the sporulation process, plcR transcription, and consequently PlcR-regulated gene expression, is repressed by the sporulation key-regulator Spo0A. Full article ">Figure 4
Molecular model of the B. cereus haemolysin II heptameric pore. Molecular modeling was performed using the 3D structure of α-hemolysin from S. aureus [<a href="#B60-toxins-05-01119" class="html-bibr">60</a>]. Full article ">Figure 5
Model of the role and expression of hlyII during infection (A) As long as iron and glucose are abundant in the bacterial environment, glucose enters the bacteria as glucose 6P (blue rectangles) and binds HlyIIR (plain orange cross). Iron (purple circles) binds Fur (red ovals). These binding events promote the repressor activities of HlyIIR and Fur, leading to the HlyIIR- and Fur-based transcriptional repression of hlyII gene expression (B) By contrast, when glucose and iron become scarce, hlyII expression is activated. HlyII is then released into the environment and induces macrophage and erythrocyte lysis. The dead cells release their intracellular content, providing access to metabolites that are essential for bacterial growth [<a href="#B75-toxins-05-01119" class="html-bibr">75</a>]. Full article ">

894 KiB

Open AccessArticle

Reduction of Streptolysin O (SLO) Pore-Forming Activity Enhances Inflammasome Activation

by Peter A. Keyel, Robyn Roth, Wayne M. Yokoyama, John E. Heuser and Russell D. Salter

Toxins 2013, 5(6), 1105-1118; https://doi.org/10.3390/toxins5061105 - 6 Jun 2013

Cited by 44 | Viewed by 9789

Abstract

Pore-forming toxins are utilized by bacterial and mammalian cells to exert pathogenic effects and induce cell lysis. In addition to rapid plasma membrane repair, macrophages respond to pore-forming toxins through activation of the NLRP3 inflammasome, leading to IL-1? secretion and pyroptosis. The structural [...] Read more.

Pore-forming toxins are utilized by bacterial and mammalian cells to exert pathogenic effects and induce cell lysis. In addition to rapid plasma membrane repair, macrophages respond to pore-forming toxins through activation of the NLRP3 inflammasome, leading to IL-1? secretion and pyroptosis. The structural determinants of pore-forming toxins required for NLRP3 activation remain unknown. Here, we demonstrate using streptolysin O (SLO) that pore-formation controls IL-1? secretion and direct toxicity. An SLO mutant incapable of pore-formation did not promote direct killing, pyroptosis or IL-1? production. This indicated that pore formation is necessary for inflammasome activation. However, a partially active mutant (SLO N402C) that was less toxic to macrophages than wild-type SLO, even at concentrations that directly lysed an equivalent number of red blood cells, enhanced IL-1? production but did not alter pyroptosis. This suggests that direct lysis may attenuate immune responses by preventing macrophages from successfully repairing their plasma membrane and elaborating more robust cytokine production. We suggest that mutagenesis of pore-forming toxins represents a strategy to enhance adjuvant activity. Full article

(This article belongs to the Special Issue Pore-Forming Toxins)

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Pore formation of streptolysin O (SLO) mutants. Blebbing was induced (A,B,D–G), or not induced (C) in CHO cells by treatment with 25 mM paraformaldehyde and 2 mM DTT. Subsequently, no SLO (A), 10,000 U/mL SLO C530A (B,D,E) or an equivalent mass of SLO N402E (F) or SLO N402C (G,H) were added to the cells, and the cells were prepared for EM. Alternatively, living CHO cells were treated with 125 U/mL SLO for 5 min (C) and prepared for EM. Arrowheads indicate strands formed by SLO N402C. Scale bar = 1 μm (A,B) or 50 nm (C–H). Full article ">Figure 2
SLO N402C shows reduced direct toxicity. LPS-primed bone marrow-derived macrophages (BMDM)from B6 or Casp1−/− mice were treated with the indicated SLO mutants at the indicated concentrations for 5 min (A,B) or 30 min (C,D) and PI uptake analyzed by FACS. The percentage of PIhigh, or dead cells (A,C) or percentage of PIl°w, or transiently permeabilized cells (B,D) are shown. The graphs display the average ± SEM of 3 independent experiments. * indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001 for comparisons between wild type and N402C SLO (A,B) or comparisons between B6 and Casp1−/− BMDM (C,D). Full article ">Figure 3
SLO C530A/N402C also shows reduced toxicity. LPS-primed BMDM were treated with the indicated SLO mutants at the indicated concentrations (1000 U/mL for C) for 5 min or 30 min in the absence (A–C) or presence (C) of 50 mM KCl and PI uptake analyzed by FACS. The percentage of PIhigh, or dead cells (A,C) or percentage of PIl°w, or transiently permeabilized cells (B,C) are shown. The graphs display the average ± SEM of 3 independent experiments. * indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001. Full article ">Figure 4
SLO N402C enhances IL-1β release. (A–E) LPS-primed B6 or Casp1−/− BMDM were treated with the indicated inhibitors and concentrations of SLO mutants for 30 min, or the controls nigericin or nothing. Supernatants were harvested and assayed for IL-1β levels by ELISA. (F) LPS-primed BMDM were treated as indicated with controls or 2000 U/mL of each SLO mutant for 30 min. Supernatants were collected and TCA precipitated, while cells were lysed in SDS-sample buffer. Lysates and supernatants were resolved by SDS-PAGE, transferred to PVDF and probed with anti-IL-1β monoclonal antibody 3ZD and anti-actin monoclonal antibody. The graphs display the average of 4 independent experiments ± SEM, while the blot shows one representative experiment of 5. * indicates p < 0.05 and ** indicates p < 0.01. Full article ">

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Open AccessArticle

Limited Stability of Microcystins in Oligopeptide Compositions of Microcystis aeruginosa (Cyanobacteria): Implications in the Definition of Chemotypes

by Ramsy Agha, Samuel Cirés, Lars Wörmer and Antonio Quesada

Toxins 2013, 5(6), 1089-1104; https://doi.org/10.3390/toxins5061089 - 6 Jun 2013

Cited by 16 | Viewed by 10093

Abstract

The occurrence of diverse oligopeptides in cyanobacteria, including the cyanotoxins microcystins, has been recently used to classify individual clones into sub-specific oligopeptide chemotypes, whose composition and dynamics modulate microcystin concentrations in cyanobacterial blooms. Cyanobacterial chemotyping allows the study of the ecology of chemotypical [...] Read more.

The occurrence of diverse oligopeptides in cyanobacteria, including the cyanotoxins microcystins, has been recently used to classify individual clones into sub-specific oligopeptide chemotypes, whose composition and dynamics modulate microcystin concentrations in cyanobacterial blooms. Cyanobacterial chemotyping allows the study of the ecology of chemotypical subpopulations, which have been shown to possess dissimilar ecological traits. However, the stability of chemotypes under changing abiotic conditions is usually assumed and has not been assessed in detail. We monitored oligopeptide patterns of three strains of Microcystis aeruginosa under different nutrient and light conditions. MALDI-TOF MS revealed alterations in the microcystins signatures under N and P poor conditions and high light intensities (150 and 400 ?mol photons m^?2s^?1). Variations in the general oligopeptide composition were caused by a gradual disappearance of microcystins with low relative intensity signals from the fingerprint. The extent of such variations seems to be closely related to physiological stress caused by treatments. Under identical clonal compositions, alterations in the oligopeptide fingerprint may be misinterpreted as apparent shifts in chemotype succession. We discuss the nature of such variations, as well as the consequent implications in the use of cyanobacterial chemotyping in studies at the subpopulation level and propose new guidance for the definition of chemotypes as a consistent subpopulation marker. Full article

(This article belongs to the Special Issue Cyanotoxins)

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384 KiB

Open AccessArticle

Currencies of Mutualisms: Sources of Alkaloid Genes in Vertically Transmitted Epichloae

by Christopher L. Schardl, Carolyn A. Young, Juan Pan, Simona Florea, Johanna E. Takach, Daniel G. Panaccione, Mark L. Farman, Jennifer S. Webb, Jolanta Jaromczyk, Nikki D. Charlton, Padmaja Nagabhyru, Li Chen, Chong Shi and Adrian Leuchtmann

Toxins 2013, 5(6), 1064-1088; https://doi.org/10.3390/toxins5061064 - 6 Jun 2013

Cited by 109 | Viewed by 13170

Abstract

The epichloae (Epichloë and Neotyphodium species), a monophyletic group of fungi in the family Clavicipitaceae, are systemic symbionts of cool-season grasses (Poaceae subfamily Poöideae). Most epichloae are vertically transmitted in seeds (endophytes), and most produce alkaloids that attack nervous systems of potential [...] Read more.

The epichloae (Epichloë and Neotyphodium species), a monophyletic group of fungi in the family Clavicipitaceae, are systemic symbionts of cool-season grasses (Poaceae subfamily Poöideae). Most epichloae are vertically transmitted in seeds (endophytes), and most produce alkaloids that attack nervous systems of potential herbivores. These protective metabolites include ergot alkaloids and indole-diterpenes (tremorgens), which are active in vertebrate systems, and lolines and peramine, which are more specific against invertebrates. Several Epichloë species have been described which are sexual and capable of horizontal transmission, and most are vertically transmissible also. Asexual epichloae are mainly or exclusively vertically transmitted, and many are interspecific hybrids with genomic contributions from two or three ancestral Epichloë species. Here we employ genome-scale analyses to investigate the origins of biosynthesis gene clusters for ergot alkaloids (EAS), indole-diterpenes (IDT), and lolines (LOL) in 12 hybrid species. In each hybrid, the alkaloid-gene and housekeeping-gene relationships were congruent. Interestingly, hybrids frequently had alkaloid clusters that were rare in their sexual ancestors. Also, in those hybrids that had multiple EAS, IDT or LOL clusters, one cluster lacked some genes, usually for late pathway steps. Possible implications of these findings for the alkaloid profiles and endophyte ecology are discussed. Full article

(This article belongs to the Special Issue Evolutionary/Phylogenetic Studies of Mycotoxin Biosynthetic Pathways 2013)

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687 KiB

Open AccessCase Report

Treatment of Cyanobacterial (Microcystin) Toxicosis Using Oral Cholestyramine: Case Report of a Dog from Montana

by Kelly A. Rankin, Karen A. Alroy, Raphael M. Kudela, Stori C. Oates, Michael J. Murray and Melissa A. Miller

Toxins 2013, 5(6), 1051-1063; https://doi.org/10.3390/toxins5061051 - 29 May 2013

Cited by 26 | Viewed by 12109

Abstract

A two and a half year old spayed female Miniature Australian Shepherd presented to a Montana veterinary clinic with acute onset of anorexia, vomiting and depression. Two days prior, the dog was exposed to an algal bloom in a community lake. Within h, [...] Read more.

A two and a half year old spayed female Miniature Australian Shepherd presented to a Montana veterinary clinic with acute onset of anorexia, vomiting and depression. Two days prior, the dog was exposed to an algal bloom in a community lake. Within h, the animal became lethargic and anorexic, and progressed to severe depression and vomiting. A complete blood count and serum chemistry panel suggested acute hepatitis, and a severe coagulopathy was noted clinically. Feces from the affected dog were positive for the cyanobacterial biotoxin, microcystin-LA (217 ppb). The dog was hospitalized for eight days. Supportive therapy consisted of fluids, mucosal protectants, vitamins, antibiotics, and nutritional supplements. On day five of hospitalization, a bile acid sequestrant, cholestyramine, was administered orally. Rapid clinical improvement was noted within 48 h of initiating oral cholestyramine therapy. At 17 days post-exposure the dog was clinically normal, and remained clinically normal at re-check, one year post-exposure. To our knowledge, this is the first report of successful treatment of canine cyanobacterial (microcystin) toxicosis. Untreated microcystin intoxication is commonly fatal, and can result in significant liver damage in surviving animals. The clinical success of this case suggests that oral administration of cholestyramine, in combination with supportive therapy, could significantly reduce hospitalization time, cost-of-care and mortality for microcystin-poisoned animals Full article

(This article belongs to the Special Issue Cyanotoxins)

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Toxins, Volume 5, Issue 6 (June 2013) – 9 articles , Pages 1051-1218

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