Diagnosing Microcystin Intoxication of Canines: Clinicopathological Indications, Pathological Characteristics, and Analytical Detection in Postmortem and Antemortem Samples

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^-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: Adda; ELISA; HAB; LC-MS/MS; MMPB; canine intoxication; hair; microcystin; urinalysis.

Figure 1

Photomicrographs (canine) of hematoxylin and eosin stained (H&E) normal liver, renal cortex, and thymus (A,C,E) as compared to the MC exposed dog (C-SP) (B,D,F). (B) Severely disrupted hepatic cords characterized by massive hepatocellular necrosis and hemorrhage (asterisk). Low numbers of hepatocytes adjacent to a central vein are spared. (D) Renal cortex with a locally extensive area of acute tubular necrosis. Note accumulation of brown granular pigment within tubular epithelial cytoplasm or sloughed cellular debris within the tubular lumina (arrow). (F) Mediastinal adipose and thymus expanded by hemorrhage, fibrin and edema (asterisks). Scale bars are 100, 50, and 500 µm for liver, renal cortex and thymus, respectively.

Figure 2

A Microcystis colony observed in the canine vomitus sample acquired within 6 h of exposure. The micrographs are at 400× with brightfield (top), phase-contrast (middle) and epi-fluorescence (bottom). The scale bar represents 25 µm.

Figure 3

The LC-MS/MS chromatograms of MC variants confirmed present in the C-GR#2 vomit sample with a sum of 14,000 ng g⁻¹ MCs. The MC-LR scale is on the right due to high levels detected in comparison to the other variants. MC-LR > [Dha⁷]MC-LR > MC-HilR > [DAsp³]MC-LR > MC-LY > MC-LW > MC-LF. Transitions monitored are reported in Table S1.

Figure 4

Data derived from the log of total Adda MCs (by MMPB) of the urine collected from one of the surviving dogs (C-GR#2) plotted against days post exposure. Urine was collected within 1 day from initial exposure event, and the animal continued to excrete MC metabolites >60 days post exposure. The MDL for total MCs in urine was determined to be 0.2 ng mL⁻¹.

Figure 5

MMPB LC-MS/MS chromatograms (m/z 207→131) showing sample peaks (blue) overlaid with their paired pre-oxidation MC-LR spikes (red) at 200 ng g⁻¹ for A (exposed dog C-GR#1) and 100 ng-g⁻¹ for B (unexposed dog C-CBR). Total MCs detected in the C-GR#1 hair at 72 days post exposure was determined to be 180 ng g⁻¹, illustrating hair as a potential route of MC elimination.

Figure 6

Results showing the different specimens collected from the dog (C-SP) that succumbed to intoxication 2 days post exposure and reported as ppb (ng mL⁻¹ or ng g⁻¹). As illustrated, the urine contained the highest amounts of MCs (all methods), followed with the bile, kidney, liver and finally heart blood. All specimens were collected post-mortem.

Figure 7

Schematic showing the recommended approach to the oxidation, extraction and clean-up of urine for the purposes of total Adda MCs by the MMPB method. In order to properly quantitate, it is essential that pre-oxidation spiking of MC-LR be conducted on duplicate sample.