**Amanda J. Foss 1,\*, Mark T. Aubel 1, Brandi Gallagher 2, Nancy Mettee 3, Amanda Miller <sup>3</sup> and Susan B. Fogelson <sup>4</sup>**


Received: 13 June 2019; Accepted: 1 August 2019; Published: 3 August 2019

**Abstract:** 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<sup>−</sup>1) 1-day post exposure, with MCs detectable >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.

**Keywords:** HAB; microcystin; Adda; canine intoxication; MMPB; urinalysis; hair; ELISA; LC-MS/MS

**Key Contribution:** Multi-modal (clinal; pathological; analytical) investigation of an acute microcystin exposure event led to the discovery of ideal specimens for postmortem (kidney)and antemortem (urine) testing. The recommended method for detection; the MMPB technique; provided low level detection of total Adda MCs/NODs in urine and hair; which has not been previously described.
