Dwayne E. Schrunk

Ergot alkaloid intoxication in perennial ryegrass (Lolium perenne): an emerging animal health concern in Ireland?

Four primary mycotoxicosis have been reported in livestock caused by fungal infections of grasses or cereals by members of the Clavicipitaceae family. Ergotism (generally associated with grasses, rye, triticale and other grains) and fescue toxicosis (associated with tall fescue grass, Festuca arundinacea) are both caused by ergot alkaloids, and referred to as ‘ergot alkaloid intoxication’. Ryegrass staggers (associated with perennial ryegrass Lolium perenne) is due to intoxication with an indole-diperpene, Lolitrem B, and metabolites. Fescue-associated oedema, recently described in Australia, may be associated with a pyrrolizidine alkaloid, N-acetyl norloline.Ergotism, caused by the fungus Claviceps purpurea, is visible and infects the outside of the plant seed. Fescue toxicosis and ryegrass staggers are caused by Neotyphodium coenophalium and N. lolii, respectively. Fescue-associated oedema has been associated with tall fescue varieties infected with a specific strain of N. coenophialum (AR542, Max P or Max Q). The name Neotyphodium refers to asexual derivatives of Epichloë spp., which have collectively been termed the epichloë fungi. These fungi exist symbiotically within the grass and are invisible to the naked eye.The primary toxicological effect of ergot alkaloid involves vasoconstriction and/or hypoprolactinaemia. Ingestion of ergot alkaloid by livestock can cause a range of effects, including poor weight gain, reduced fertility, hyperthermia, convulsions, gangrene of the extremities, and death. To date there are no published reports, either internationally or nationally, reporting ergot alkaloid intoxication specifically associated with perennial ryegrass endophytes. However, unpublished reports from the Irish Equine Centre have identified a potential emerging problem of ergot alkaloid intoxication with respect to equines and bovines, on primarily perennial ryegrass-based diets. Ergovaline has been isolated in varying concentrations in the herbage of a small number of equine and bovine farms where poor animal health and performance had been reported. Additionally, in some circumstances changes to the diet, where animals were fed primarily herbage, were sufficient to reverse adverse effects. Pending additional information, these results suggest that Irish farm advisors and veterinarians should be aware of the potential adverse role on animal health and performance of ergot alkaloids from perennial ryegrass infected with endophytic fungi.

Quality of veterinary pharmaceuticals and their use by pastoralists in the Far North Region of Cameroon

This study evaluates the quality of the veterinary drugs most frequently used in the Far North Region of Cameroon and describes how pastoralists use them to treat their cattle herds. High-performance liquid chromatography was used to identify and quantify the active ingredients in the drugs (penicillin G, levamisole, oxytetracycline, diminazene diaceturate, vitamin A, and vitamin E acetate) and gas chromatography mass spectrometry to determine if organic chemical contaminants were present. The results showed that 69% of surveyed pastoralists used veterinary medicines to treat common illnesses. In addition, the most commonly used medications (procaine penicillin G and oxytetracycline) were used in a manner inconsistent with the recommended dosage, frequency, duration, and withdrawal period by 98% of the pastoralists. However, contrary to previous studies, the quality of the medications used by pastoralists was generally good. The poor compliance with recommended treatment protocols was much more prevalent than use of poor quality medications and presents a potential for treatment failure, drug resistance of animal pathogens, and harmful drug residues in the human food supply, all of which have potentially negative consequences for animal and human health.

Mycotoxin and metallic element concentrations in peanut products sold in Ugandan markets

Abstract The increasing prevalence of cancer among Ugandans has aroused consumer concerns about food-borne carcinogens. This study sought to compare mycotoxin and metallic element concentrations in processed peanuts sold in selected markets in Kampala, Uganda to those traditionally prepared in homes. Market-processed peanut samples (n = 33) were purchased from four markets. Control samples (n = 5) were unground peanuts bought from markets but processed in homes by traditional methods. Aflatoxins B1, B2, G1, G2; Fumonisins; Deoxynivalenol, Nivalenol, Ochratoxin A, T2 toxin, Zearalenone, and Zearalenol were analyzed by LC/MS/MS while As, B, Ba, Cd, Cr, Cu, Hg, Mg, Ni, Pb and Zn were analyzed by ICP/MS. The data was statistically analyzed using Wilcoxon scores (rank sums) or the Kruskal-Wallis test. Aflatoxins were the predominant mycotoxins found in significant amounts. 55 and 34% of the samples had concentrations of total aflatoxins greater than 20 ppb (FDA acceptable limit). There were significantly higher concentrations of aflatoxins in market-processed than in home-processed samples. Metallic element concentrations were generally below FDA maximum acceptable concentrations. Roasting and duration of grinding had no significant effect on aflatoxin or metallic element concentrations. There is a need for food-borne toxicant monitoring of food sold in public markets in Uganda.

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.

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.

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.