Intra-laboratory Development and Evaluation of a Quantitative Method for Measurement of Aflatoxins B1, M1 and Q1 in Animal Urine by High Performance Liquid Chromatography with Fluorescence Detection.

Mycotoxins negatively impact animal health. Aflatoxins (AFs) are the most common mycotoxins affecting both large and small animals and are a common cause of toxin-related pet food recalls. Definitive diagnosis of aflatoxicosis is constrained by a lack of validated ante-mortem analytical methods for detection and quantitation of AFs and their metabolites in biological specimens. Herein, we developed and evaluated a urine-based quantitative method for measurement of aflatoxin B1 (AFB1) and its metabolites aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1) in animal urine. (Some of the results have been presented at 59th AAVLD conference, Greensboro, North Carolina, October 13-19th, 2016.) This method uses an immuno-affinity column for clean-up and pre-column derivatization followed by high performance liquid chromatography analysis with fluorescence detection. The method has high selectivity, recovery (>81%) and sensitivity with an instrument limit of detection of 0.20-1.02 pg; instrument limit of quantitation of 0.77-4.46 pg; and a method lower limit of quantitation of 0.30-2.5 ng/mL. The method has high accuracy, repeatability, and is rugged against minor changes. However, because of poor sensitivity of AFQ1 at low concentrations we recommend this method for quantitative determination of AFB1 and AFM1, and for qualitative measurement of AFQ1 in animal urine for diagnosis of aflatoxicosis.

Characterizing a mouse model for evaluation of countermeasures against hydrogen sulfide-induced neurotoxicity and neurological sequelae.

Hydrogen sulfide (H2 S) is a highly neurotoxic gas. It is the second most common cause of gas-induced deaths. Beyond mortality, surviving victims of acute exposure may suffer long-term neurological sequelae. There is a need to develop countermeasures against H2 S poisoning. However, no translational animal model of H2 S-induced neurological sequelae exists. Here, we describe a novel mouse model of H2 S-induced neurotoxicity for translational research. In paradigm I, C57/BL6 mice were exposed to 765 ppm H2 S for 40 min on day 1, followed by 15-min daily exposures for periods ranging from 1 to 6 days. In paradigm II, mice were exposed once to 1000 ppm H2 S for 60 minutes. Mice were assessed for behavioral, neurochemical, biochemical, and histopathological changes. H2 S intoxication caused seizures, dyspnea, respiratory depression, knockdowns, and death. H2 S-exposed mice showed significant impairment in locomotor and coordinated motor movement activity compared with controls. Histopathology revealed neurodegenerative lesions in the collicular, thalamic, and cortical brain regions. H2 S significantly increased dopamine and serotonin concentration in several brain regions and caused time-dependent decreases in GABA and glutamate concentrations. Furthermore, H2 S significantly suppressed cytochrome c oxidase activity and caused significant loss in body weight. Overall, male mice were more sensitive than females. This novel translational mouse model of H2 S-induced neurotoxicity is reliable, reproducible, and recapitulates acute H2 S poisoning in humans.

Intralaboratory development and evaluation of a high-performance liquid chromatography-fluorescence method for detection and quantitation of aflatoxins M1, B1, B2, G1, and G2 in animal liver.

Aflatoxins are potent mycotoxins with effects that include hepatotoxicity, immunosuppression, and suppression of animal growth and production. The etiologic diagnosis of aflatoxicosis, which is largely based on analysis of contaminated feed matrices, has significant disadvantages given the fact that representative feed samples may not be available and feed-based test methods are not confirmatory of an etiologic diagnosis. A tissue-based analytical method for biomarkers of exposure would be valuable for confirmation of aflatoxicosis. We describe in-house development and evaluation of a high-performance liquid chromatographic method with fluorescence detection and precolumn derivatization for determination of aflatoxins M1, B1, B2, G1, and G2 in animal liver. The method demonstrates good selectivity for the tested aflatoxins in the liver matrix. The overall range was 0.03-0.10 ng/g for limit of detection and 0.09-0.18 ng/g for limit of quantitation. The correlation coefficient (R2) of calibration curves was >0.9978 for AFM1, 0.9995 for AFB1, 0.9986 for AFB2, 0.9983 for AFG1, and 0.9980 for AFG2 For fortification levels of 0.2-10 ng/g, repeatability was 10-18% for AFM1, 7-14% for AFB1, 5-14% for AFB2, 6-16% for AFG1, and 10-15% for AFG2 Recovery was 52-57% for AFM1, 54-62% for AFB1, 55-61% for AFB2, 57-67% for AFG1, and 61-65% for AFG2 There was no liver matrix effect found. The method is rugged against minor changes based on the selected factors. The results indicate that the proposed method is suitable for quantitative determination of aflatoxins M1, B1, B2, G1, and G2 in liver.

Improved Tissue-Based Analytical Test Methods for Orellanine, a Biomarker of Cortinarius Mushroom Intoxication.

Orellanine (OR) toxin is produced by mushrooms of the genus Cortinarius which grow in North America and in Europe. OR poisoning is characterized by severe oliguric acute renal failure, with a mortality rate of 10%-30%. Diagnosis of OR poisoning currently hinges on a history of ingestion of Cortinarius mushrooms and histopathology of renal biopsies. A key step in the diagnostic approach is analysis of tissues for OR. Currently, tissue-based analytical methods for OR are nonspecific and lack sensitivity. The objectives of this study were: (1) to develop definitive HPLC and LC-MS/MS tissue-based analytical methods for OR; and (2) to investigate toxicological effects of OR in mice. The HPLC limit of quantitation was 10 µg/g. For fortification levels of 15 µg/g to 50 µg/g OR in kidney, the relative standard deviation was between 1.3% and 9.8%, and accuracy was within 1.5% to 7.1%. A matrix-matched calibration curve was reproduced in this range with a correlation coefficient (r) of 0.97-0.99. The limit of detection was 20 ng/g for LC-MS/MS. In OR-injected mice, kidney OR concentrations were 97 ± 51 µg/g on Day 0 and 17 ± 1 µg/g on termination Day 3. Splenic and liver injuries were novel findings in this mouse model. The new tissue-based analytical tests will improve diagnosis of OR poisoning, while the mouse model has yielded new data advancing knowledge on OR-induced pathology. The new tissue-based analytical tests will improve diagnosis of OR poisoning, while the mouse model has yielded new data advancing knowledge on OR-induced pathology.

A novel orellanine containing mushroom Cortinarius armillatus.

Orellanine (3,3',4,4'-tetrahydroxy-2,2'-bipyridine-1,1'-dioxide) is a tetrahydroxylated di-N-oxidized bipyridine compound. The toxin, present in certain species of Cortinarius mushrooms, is structurally similar to herbicides Paraquat and Diquat. Cortinarius orellanus and Cortinarius rubellus are the major orellanine-containing mushrooms. Cortinarius mushrooms are widely reported in Europe where they have caused human poisoning and deaths through accidental ingestion of the poisonous species mistaken for the edible ones. In North America, Cortinarius orellanosus mushroom poisoning was recently reported to cause renal failure in a Michigan patient. Cortinarius mushroom poisoning is characterized by delayed acute renal failure, with some cases progressing to end-stage kidney disease. There is debate whether other Cortinarius mushroom contain orellanine or not, especially in North America. Currently, there are no veterinary diagnostic laboratories in North America with established test methods for detection and quantitation of orellanine. We have developed two diagnostic test methods based on HPLC and LC-MSMS for identification and quantitation of orellanine in mushrooms. Using these methods, we have identified Cortinarius armillatus as a novel orellanine-containing mushroom in North America. The mean toxin concentration of 145 ug/g was <1% of that of the more toxic C. rubellus. The HPLC method can detect orellanine at 17 µg g(-1) while the LC-MSMS method is almost 2000 times more sensitive and can detect orellanine at 30 ng g(-1). Both tests are quantitative, selective and are now available for veterinary diagnostic applications.