Search

Microcystins (MCs) and nodularins (NODs) exhibit high structural variability, including modifications of the Adda (3S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid) moiety. Variations include 9-O-desmethylAdda (DMAdda) and 9-O-acetylDMAdda (ADMAdda) which, unless targeted, may go undetected. Therefore, reference standards were prepared of [ADMAdda5]MCs and [DMAdda5]MCs, which were analyzed using multiple approaches. The cross-reactivities of the [DMAdda5]- and [ADMAdda5]MC standards were similar to that of MC-LR when analyzed with a protein phosphatase 2A (PP2A) inhibition assay, but were, Graphical abstract Image 1, Highlights • Toxic ADMAdda/DMAdda microcystins (MCs) exhibit low cross-reactivity with Adda ELISA. • A method for oxidative cleavage was expanded to test for ADMAdda/DMAdda MCs. • Two sites of oxidative cleavage of the Adda/DMAdda/ADMAdda were observed. • Oxidation products were identified and analytically tested to quantitate ‘total MCs’.

In the summer of 2012, over 750 dead and dying birds were observed at the Paul S. Sarbanes Ecosystem Restoration Project at Poplar Island, Maryland, USA (Chesapeake Bay). Clinical signs suggested avian botulism, but an ongoing dense Microcystis bloom was present in an impoundment on the island. Enzyme-linked immunosorbent assay (ELISA) analysis of a water sample indicated 6000 ng mL −1 of microcystins (MCs). LC-UV/MS analysis confirmed the presence of MC-LR and a high concentration of an unknown MC congener ( m/z 1037.5). The unknown MC was purified and confirmed to be [D-Leu 1 ]MC-LR using NMR spectroscopy, LC-HRMS and LC–MS 2 , which slowly converted to [D-Leu 1 ,Glu(OMe) 6 ]MC-LR during storage in MeOH. Lyophilized algal material from the bloom was further characterized using LC-HRMS and LC–MS 2 in combination with chemical derivatizations, and an additional 24 variants were detected, including MCs conjugated to Cys, GSH and γ-GluCys and their corresponding sulfoxides. Mallard ( Anas platyrhynchos ) livers were tested to confirm MC exposure. Two broad-specificity MC ELISAs and LC–MS 2 were used to measure free MCs, while ‘total’ MCs were estimated by both MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) and thiol de-conjugation techniques. Free microcystins in the livers (63–112 ng g −1 ) accounted for 33–41% of total microcystins detected by de-conjugation and MMPB techniques. Free [D-Leu 1 ]MC-LR was quantitated in tissues at 25–67 ng g −1 (LC–MS 2 ). The levels of microcystin varied based on analytical method used, highlighting the need to develop a comprehensive analysis strategy to elucidate the etiology of bird mortality events when microcystin-producing HABs are present.

In the summer of 2018, six dogs exposed to a harmful algal bloom (HAB) of Microcystis in Martin County Florida (USA) developed clinicopathological signs of microcystin (MC) intoxication (i.e., acute vomiting, diarrhea, severe thrombocytopenia, elevated alanine aminotransferase, hemorrhage). Successful supportive veterinary care was provided and led to survival of all but one patient. Confirmation of MC intoxication was made through interpretation of clinicopathological abnormalities, pathological examination of tissues, microscopy (vomitus), and analytical MC testing of antemortem/postmortem samples (vomitus, blood, urine, bile, liver, kidney, hair). Gross and microscopic examination of the deceased patient confirmed massive hepatic necrosis, mild multifocal renal tubular necrosis, and hemorrhage within multiple organ systems. Microscopy of a vomitus sample confirmed the presence of Microcystis. Three analytical MC testing approaches were used, including the MMPB (2-methyl-3-methoxy-4-phenylbutyric acid) technique, targeted congener analysis (e.g., liquid chromatography tandem-mass spectrometry of MC-LR), and enzyme-linked immunosorbent assay (ELISA). Total Adda MCs (as MMPB) were confirmed in the liver, bile, kidney, urine, and blood of the deceased dog. Urinalysis (MMPB) of one surviving dog showed a high level of MCs (32,000 ng mL&minus, 1) 1-day post exposure, with MCs detectable &gt, 2 months post exposure. Furthermore, hair from a surviving dog was positive for MMPB, illustrating another testable route of MC elimination in canines. The described cases represent the first use of urine as an antemortem, non-invasive specimen to diagnose microcystin toxicosis. Antemortem diagnostic testing to confirm MC intoxication cases, whether acute or chronic, is crucial for providing optimal supportive care and mitigating MC exposure.

Algal samples were analyzed from 3 lakes, Crane Prairie Reservoir and Odell Lake in Oregon and an Anonymous North East System, using both standard taxonomic criteria for identification and DNA sequencing techniques. Two toxin-producing Anabaena populations, one with consistent akinete structure and another with variable akinete structure, were investigated. Samples were characterized based on several genetic markers (nifH, cpcBA-IGS, ITS1), toxins (anatoxin-a, saxitoxin, and microcystin) and morphological variation. Taxonomy within the Nostacales is based on vegetative and terminal cell structure, filament type and aggregation, and position and structure of heterocysts and akinetes. Many taxonomists rely heavily on akinete structure for microscopic identification. Identification from material preserved with Lugol's solution is challenging due to the breakup of colonies, cell distortion, and masking of pigment color. Based on morphological variation, the Crane Prairie and Odell populations were id...

Microcystin poisoning was diagnosed in a dog exposed to a Microcystis aeruginosa-dominated, freshwater, harmful algal bloom at Milford Lake, Kansas, which occurred during the summer of 2011. Lake water microcystin concentrations were determined at intervals during the summer, using competitive enzyme-linked immunosorbent assays, and indicated extremely high, localized microcystin concentrations of up to 126,000 ng/ml. Multiple extraction and analysis techniques were used in the determination of free and total microcystins in vomitus and liver samples from the poisoned dog. Vomitus and liver contained microcystins, as determined by enzyme-linked immunosorbent assays, and the presence of microcystin-LR was confirmed in vomitus and liver samples using liquid chromatography coupled with tandem mass spectrometry. Major toxic effects in a dog presented for treatment on the day following exposure included fulminant liver failure and coagulopathy. The patient deteriorated rapidly despite aggressive treatment and was euthanized. Postmortem lesions included diffuse, acute, massive hepatic necrosis and hemorrhage, as well as acute necrosis of the renal tubular epithelium. A diagnosis of microcystin poisoning was based on the demonstration of M. aeruginosa and microcystin-LR in the lake water, as well as in vomitus produced early in the course of the poisoning; the presence of microcystin-LR in liver tissue; and a typical clinical course including gastroenteritis and fulminant liver failure.

The eutrophication of water resources, mainly attributed to antrophogenic activities such as sewage and agricultural runoffs, has led to a worldwide increase in the formation of cyanobacterial harmful algal blooms (Cyano-HABs). Cyano-HABs have the ability to produce and release toxic compounds, commonly known as cyanotoxins, which comprise a potent threat for human and animal health as well as negative economical impacts. This chapter presents an overview on the sources and occurrence of species of cyanobacteria and their association with the production of cyanotoxins throughout the world. The main bloom-forming cyanobacteria that have been detected include Microcystis, Cylindrospermopsis, Anabaena, Aphanizomenon, and Planktothrix. The main cyanotoxins related to these cyanobacteria are microcystins, cylindrospermopsin, anatoxin-a and saxitoxins.