Ergot Alkaloid Concentrations Research Articles

Mycotoxins in vegetating corn plants from experimental mono-sowing

The aim of present study was to reveal the mycotoxin contamination of vegetative maize (Zea mays L.) plants during the periods of leaf formation and panicle emergence. Early maturing hybrids of the varieties Krasnodarsky 194 MV, Ladozhsky 175 MV and Competence®, resistant to fungal diseases, were grown in the spring-summer period of 2023 on the experimental field of the Russian State Agrarian University – Moscow Agricultural Academy (Moscow) with sod-podzolic soil and the application of NPK fertilizers 16:16:16. For mycotoxicological analysis, aerial parts of plants were collected weekly from the phase of formation of the 3rd leaf (18 days after sowing. Samples of seedlings, leaves and stems (total number – 172), after drying, were ground in a laboratory mill and extracted with a mixture of acetonitrile and water in a volumetric ratio of 84:16 with a consumption of 10 ml per 1 g of sample. Mycotoxin content was determined in extracts after 10-fold dilution with phosphate-salt buffer solution pH 7.5 by indirect competitive enzyme immunoassay. T-2 toxin, deoxynivalenol, diacetoxiscirpenol, roridin A, sterigmatocystin and PR toxin were absent in the samples. Corn seedlings and leaves contained cyclopiazonic acid (CPA), emodin (EMO), mycophenolic acid (MPA), alternariol (AOL), ergot alkaloids (EA), aflatoxin B1 (AB1) and single samples – zearalenone (ZEN), ochratoxin A (OA) and citrinin (CIT) in concentrations from 16 up to 35 μg/kg. Fumonisins of group B are found only in early seedlings. The permanent contaminant of the stems was MPA, whereas EA and CIT were absent. In the seedlings and leaves of all hybrids, the detection of AOL and EA remained stable during the change of development phases, as did the average concentrations of CPA (about 100 μg/kg), AOL (from 17 to 27 μg/kg), EMO (35–58 μg/kg), MPA (28–41 μg/kg), EA (6–18 μg/kg) and AB1 (2 μg/kg). In the stems of plants in phases 7–9 of leaf and sweeping, variation in cases of detection of CPA, EMO, AOL, AB1 and OA was noted by varieties.

Development, in-house validation and application of a method using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) for the quantification of 12 ergot alkaloids in compound feeds.

Ergot alkaloids (EAs) are toxic substances naturally produced by Claviceps fungi. These fungi infest a wide range of cereals and grasses. When domestic animals are exposed to EAs through contaminated feeds, it is detrimental to them and leads to significant economic losses. For that reason, it is important to monitor feed for the presence of EAs, especially with methods enabling their determination in processed materials. Ergot alkaloids were extracted with acetonitrile, and dispersive solid phase extraction (d-SPE) was used for clean-up of the extracts. After evaporation, the extracts were reconstituted in ammonium carbonate and acetonitrile and subjected to instrumental analysis using high-performance liquid chromatography with fluorescence detection. The developed method was validated in terms of linearity, selectivity, repeatability, reproducibility, robustness, matrix effect, limits of quantification and detection and uncertainty. The EA content of 40 compound feeds was determined. All the assessed validation parameters fulfilled the requirements of Regulation (EU) 2021/808. At least one of the monitored alkaloids was determined in 40% of the samples. The EAs with the highest incidence rate were ergocryptine, ergometrinine and ergocornine. The total concentrations of EAs ranged from under the limit of quantification to 62.3 μg kg-1. The results demonstrated that the developed method was suitable for simultaneously determining twelve EAs in compound feed and could be used for routine analysis.

164 Effect of Processing Method of Hybrid rye on Growth Performance, Efficiency of Dietary net Energy Utilization, and Carcass Characteristics of Yearling Steers fed a Finishing Diet

Abstract Predominantly Angus steers were used in a 147-day randomized complete block design experiment (RCBD) to evaluate the effects of rye processing method on growth performance, feed efficiency, and carcass characteristics. Steers [n = 192, initial body weight (BW) = 410 ± 20.9 kg] were individually weighed and allotted to 1 of 24 pens (n = 8 steers/pen; 6 pens/treatment). Four treatments were used where hybrid rye grain (total ergot alkaloid concentration = 1,007 ppb, DM basis) was processed using three different methods [unprocessed (UP); dry-rolled (DR); or hammer-milled (HM)] fed at 17% of diet DM along with 43% dry-rolled corn (DRC) compared with a control diet containing 60% DRC on a DM basis (CON). Data were analyzed as an RCBD using the GLIMMIX procedure of SAS with treatment as the fixed effect and block (Pen location) as the random effect; pen was the experimental unit. No differences (P ≥ 0.44) were observed among treatments for live final BW or average daily gain (ADG). Dry matter intake (DMI) was greater for HM (12.27 kg) compared with CON and DR (11.84 and 11.48 kg, respectively) with DR having 4.9% less DMI compared with UP (P = 0.01; SEM = 0.200). UP and HM treatments tended to have 4.8% poorer G:F compared with CON and DR (P = 0.07). Dietary treatments did not affect carcass traits, percent empty body fatness (EBF), or final BW at 28% EBF (P ≥ 0.23). Distributions of USDA Quality or Yield grades were unaffected by treatment (P ≥ 0.23), nor was the incidence or severity of liver abscesses (P = 0.25). Estimated NEm and NEg values of the diet for UP (1.90 and 1.26 Mcal/kg, respectively) were reduced compared with CON and DR (1.98 and 1.32 Mcal/kg, respectively), with HM intermediate (P = 0.05, SEM = 0.033 and 0.029 for NEm and NEg, respectively). Net energy values for UP and HM rye were within 7% of current feeding standards when calculated using the substitution method. Rolling increased apparent ingredient NEg for rye by 30.1% compared with UP or HM rye. Processing rye grain by dry-rolling increased dietary net energy values and tended to improve gain efficiency compared with not processing or using a hammer mill. Rye grain can be partially substituted for DRC with negligible impact on growth performance or carcass measures.

The Impact of Storage Temperature and Time on Ergot Alkaloid Concentrations.

Ergot sclerotia produce toxic secondary metabolites, ergot alkaloids, that infect cereal crops and grasses. Ergot alkaloids have two isomeric configurations: the C-8-R-isomer (R-epimer), and the C-8-S-isomer (S-epimer). Ergot contaminated matrices, such as cereal grains or grasses, may be stored for extended periods at various temperatures before being analyzed, utilized, or consumed. This study assessed the concentration of six common ergot alkaloids in both configurations found in naturally contaminated wheat over time (one, two, and four months) at different temperatures (room temperature, +4 °C, and -20 °C) using ultra-high-performance liquid chromatography-tandem mass spectrometry. The data indicate that the total ergot concentration within a natural contaminated sample varies over time at room temperature, +4 °C, and -20 °C. The total ergot concentration increased until month two, and decreased at month four, independent of temperature (p < 0.05). The total R-epimer concentration appeared to be less stable over time than the total S-epimer concentration. The changes in the total R and total S-epimer concentrations may have been caused by changes in the ergocristine and ergocristinine concentrations, respectively. Time and temperature should be considered when storing potentially contaminated matrices in a laboratory or practical agriculture situations. Quantification of ergot contaminated matrices should occur prior to their use to ensure the most reliable estimates of the concentration of ergot.

89 Influence of Melatonin Supplementation in Pregnant Cattle Consuming Endophyte-Infected Tall Fescue Seed on Offspring Performance

Abstract The objective was to evaluate growth performance and estimates of body composition in offspring of dams that consumed diets containing tall fescue seed with (E+) or without (E-) the toxic endophyte and with or without melatonin during mid-late gestation. Pregnant heifers (n = 57) artificially inseminated with sex-sorted semen (X chromosome-bearing sperm) from a single Angus sire were administered dietary treatments in a 2 x 2 factorial treatment arrangement (E- without melatonin, E-/NM; E- with melatonin, E-/M; E+ without melatonin, E+/NM; or E+ with melatonin, E+/M; melatonin dose: 100 µg/kg of BW) for a 70-day period between May and July starting at 160±13 d of gestation to mimic ergot alkaloid concentrations in a grazing scenario. All diets contained corn gluten and molasses and were fed using SmartFeed Pro (C-Lock Inc.) controlled feeding units between 0900 and2 1200 h daily. Cattle were removed from dietary treatments 39 ± 19 d before calving. Two replicates were housed separately in an open-air barn, each with a 1.2-ha paddock. Prior to and following the 70-d treatment period, all cattle were maintained as a single group free from toxic fescue. Calves were weaned at 226 ± 19 d of age. Gestation length, calf birth weight, 205-day adjusted weaning weight (205-d WW), carcass ultrasonography measures (ribeye area, REA; REA/CWT; percent intramuscular fat, IMF; rib fat thickness, UFAT; rump fat thickness, URUMP), post-weaning weights (recorded every 28 d between weaning and 84 d post-weaning), and post-weaning ADG (between weaning and 84 d post-weaning) were evaluated in offspring (n = 44). Post-weaning calf weights were analyzed using the MIXED procedure of SAS specific for repeated measures with treatment and time as fixed effects and replicate as random. All other variables were analyzed using the MIXED procedure of SAS with treatment as a fixed effect and replicate as random with gestation length as a covariate for birth weight. Gestation length, birth weight, 205-d WW, and post-weaning calf weight were decreased (P &amp;lt; 0.04) in offspring of dams that consumed E+ relative to E- during gestation. Birth weight was increased (P = 0.02) in offspring of dams receiving melatonin relative to those without melatonin during gestation. The 205-d WW and REA were decreased in calves born to E+/NM dams relative to E-/NM, E-/M, and E+/M (P &amp;lt; 0.03). Post-weaning weight tended to be decreased in calves born to E+/NM dams relative to E-/NM, E-/M, and E+/M (P = 0.06). The UFAT tended to be decreased (P = 0.06) in offspring of dams that consumed E+ relative to E- during gestation. There were no other effects of treatment, time or the interaction (P &amp;gt; 0.10). To our knowledge, these preliminary results are the first to evaluate the potential of melatonin as a therapeutic to recover performance losses in offspring born to pregnant cattle consuming toxic fescue.

47 Influence of Melatonin Supplementation in Pregnant Cattle Consuming Endophyte-Infected Tall Fescue Seed on Offspring Performance

Abstract The objective was to evaluate growth performance and estimates of body composition in offspring of dams that consumed diets containing tall fescue seed with (E+) or without (E-) the toxic endophyte and with or without melatonin during mid-late gestation. Pregnant heifers (n = 57) artificially inseminated with sex-sorted semen (X chromosome-bearing sperm) from a single Angus sire were administered dietary treatments in a 2 x 2 factorial treatment arrangement (E- without melatonin, E-/NM; E- with melatonin, E-/M; E+ without melatonin, E+/NM; or E+ with melatonin, E+/M; melatonin dose: 100 µg/kg of BW) for a 70-d period between May and July starting at 160 ± 13 days of gestation to mimic ergot alkaloid concentrations in a grazing scenario. All diets contained corn gluten and molasses and were fed using SmartFeed Pro (C-Lock Inc.) controlled feeding units between 0900 and 1400 h daily. Cattle were removed from dietary treatments 39 ± 19 days before calving. Two replicates were housed separately in an open-air barn, each with a 1.2-ha paddock. before and following the 70-d treatment period, all cattle were maintained as a single group free from toxic fescue. Calves were weaned at 226 ± 19 d of age. Gestation length, calf birth weight, 205-day adjusted weaning weight (205-d WW), carcass ultrasonography measures (ribeye area, REA; REA/CWT; percent intramuscular fat, IMF; rib fat thickness, UFAT; rump fat thickness, URUMP), post-weaning weights (recorded every 28 days between weaning and 84 days post-weaning), and post-weaning ADG (between weaning and 84 days post-weaning) were evaluated in offspring (n = 44). Post-weaning calf weights were analyzed using the MIXED procedure of SAS specific for repeated measures with treatment and time as fixed effects and replicate as random. All other variables were analyzed using the MIXED procedure of SAS with treatment as a fixed effect and replicate as random with gestation length as a covariate for birth weight. Gestation length, birth weight, 205-d WW, and post-weaning calf weight were decreased (P &amp;lt; 0.04) in offspring of dams that consumed E+ relative to E- during gestation. Birth weight was increased (P = 0.02) in offspring of dams receiving melatonin relative to those without melatonin during gestation. The 205-d WW and REA were decreased in calves born to E+/NM dams relative to E-/NM, E-/M, and E+/M (P &amp;lt; 0.03). Post-weaning weight tended to be decreased in calves born to E+/NM dams relative to E-/NM, E-/M, and E+/M (P = 0.06). UFAT tended to be decreased (P = 0.06) in offspring of dams that consumed E+ relative to E- during gestation. There were no other effects of treatment, time or the interaction (P &amp;gt; 0.10). To our knowledge, these preliminary results are the first to evaluate the potential of melatonin as a therapeutic to recover performance losses in offspring born to pregnant cattle consuming toxic fescue.

Using Molecular Spectroscopic Techniques (NIR and ATR-FT/MIR) Coupling with Various Chemometrics to Test Possibility to Reveal Chemical and Molecular Response of Cool-Season Adapted Wheat Grain to Ergot Alkaloids.

The objectives of this study were to explore the possibility of using near infrared (NIR) and Fourier transform mid-infrared spectroscopy-attenuated total reflectance (ATR-FT/MIR) molecular spectroscopic techniques as non-invasive and rapid methods for the quantification of six major ergot alkaloids (EAs) in cool-season wheat. In total, 107 wheat grain samples were collected, and the concentration of six major EAs was analyzed using the liquid chromatography-tandem mass spectrometry technique. The mean content of the total EAs-ergotamine, ergosine, ergometrine, ergocryptine, ergocristine, and ergocornine-was 1099.3, 337.5, 56.9, 150.6, 142.1, 743.3, and 97.45 μg/kg, respectively. The NIR spectra were taken from 680 to 2500 nm, and the MIR spectra were recorded from 4000-700 cm-1. The spectral data were transformed by various preprocessing techniques (which included: FD: first derivative; SNV: standard normal variate; FD-SNV: first derivative + SNV; MSC: multiplicative scattering correction; SNV-Detrending: SNV + detrending; SD-SNV: second derivative + SNV; SNV-SD: SNV + first derivative); and sensitive wavelengths were selected. The partial least squares (PLS) regression models were developed for EA validation statistics. Results showed that the constructed models obtained weak calibration and cross-validation parameters, and none of the models was able to accurately predict external samples. The relatively low levels of EAs in the contaminated wheat samples might be lower than the detection limits of the NIR and ATR-FT/MIR spectroscopies. More research is needed to determine the limitations of the ATR-FT/MIR and NIR techniques for quantifying EAs in various sample matrices and to develop acceptable models.

Effect of increasing concentration of ergot alkaloids in the diet of feedlot cattle: performance, welfare, and health parameters.

This study was designed to evaluate the effects of feeding increasing dietary concentrations of ergot alkaloids from cereal grains (EA; 0, 0.75, 1.5, 3.0 mg/kg of dietary DM) to feedlot cattle over backgrounding (BG) and finishing (FS) phases on health, welfare, and growth performance. Two hundred and forty commercial steers (280 ± 32 kg BW) were stratified by weight and randomly allocated to 16 pens (15 steers/pen), 4 of which were equipped with the GrowSafe system (1 pen/treatment) to measure individual feed intake. Each pen was randomly assigned to a treatment (n = 4/treatment). Treatments included 1) control (CTRL), no added EA; 2) CTRL + 0.75 mg/kg EA (EA075); 3) CTRL + 1.5 mg/kg EA (EA150); and 4) CTRL + 3.0 mg/kg EA (EA300). Steers were fed barley-based BG diets containing 40% concentrate: 60% silage (DM basis) for 84 d. Steers were then transitioned over 28 d to an FS diet (90% concentrate: 10% silage DM basis) and fed for 119 d before slaughter. The diet fed to EA300 steers was replaced with the CTRL diet after 190 d on feed (DOF), due to EA-induced hyperthermia starting at 165 DOF. In the BG phase, average meal length (P = 0.01) and size (P = 0.02), daily feeding duration (P = 0.03), final body weight (BW; P = 0.03), and total BW gain (P = 0.02) linearly decreased with increasing EA levels, while gain to feed (G:F) responded quadratically (P = 0.04), with EA150 having the poorest value. Increasing concentrations of EA in the diet linearly increased rectal temperature (P < 0.01) throughout the trial. Over the full FS phase, a quadratic response was observed for ADG (P = 0.05), final BW (P = 0.05), total BW gain (P = 0.02), and carcass weight (P = 0.05) with steers fed EA150 having the lowest performance, as EA300 steers were transferred to CTRL diet after 190 DOF. Dressing percentage (P = 0.02) also responded quadratically, with the lowest values observed for EA300. Thus, EA reduced ADG during BG and FS phases, although more prominently in FS, likely due to increased ambient temperatures and high-energy diet in FS triggering hyperthermia. When EA300 steers were transferred to the CTRL diet, compensatory gain promoted higher hot carcass weight (HCW) when compared with steers fed EA150. In conclusion, feeding feedlot steers diets with > 0.75 mg/kg EA caused reductions in performance and welfare concerns, although this breakpoint may be affected by duration of feeding, environmental temperatures, and EA profiles in the feed.

Winter rye cultivars moderately resistant to ergot

Background. Cereal ergot caused by the fungus Claviceps purpurea (Fr.) Tul. is a progressive disease of winter rye. There are no rye cultivars resistant to the disease, and breeding methods of protection have not been developed in Russia.Materials and methods. The material for the research included 97 winter rye cultivars. A biochemical analysis of the sclerotia of the fungus was carried out, the composition and content of ergot alkaloids (EA) in the sclerotia of C. purpurea from the Kirov population were studied, and the relationship between the biometry of sclerotia and the EA content, toxicity and pathogenicity of C. purpurea was analyzed. Well-known methods of resistance assessment and EA analysis were applied.Results and conclusions. Fourteen cultivars least affected by the pathogen were identified: ‘Flora’, ‘Kiprez’, ‘Grafinya’, ‘Lika’, ‘Batist’, ‘Simfoniya’, ‘Garmoniya’, ‘Sadko’, ‘Parom’, ‘Virazh’, ‘Saratovskaya 7’, ‘Volkhova’, ‘Novaya Era’, and ‘Podarok’. They can be used in breeding for ergot resistance. Three types of EA were identified in C. purpurea sclerotia: ergocristine, ergotamine, and its stereoisomer ergocristinine. Cvs. ‘Lika’, ‘Simfoniya’ and ‘Garmoniya’ were the least affected by ergot, and the sclerotia formed on the plants of these cultivars did not accumulate EA. A significant (r = 0.50–0.60) correlation was found between lesions and biometric parameters of C. purpurea sclerotia, and between grain contamination with sclerotia and their biometrics (r = 0.63– 0.78). A weak positive correlation (r = 0.22) was detected between the toxicity and pathogenicity of C. purpurea. A negative correlation was established between the weight of sclerotia and the accumulation of EA in them (r = –0.46), which indicated the biological danger of small sclerotia that got into the seed and food batches of grain.

Effects of Heating, Pelleting, and Feed Matrix on Apparent Concentrations of Cereal Ergot Alkaloids in Relation to Growth Performance and Welfare Parameters of Backgrounding Beef Steers.

As the contamination of cereal grains with ergot has been increasing in Western Canada, studies were undertaken to evaluate the impacts of heating (60, 80, 120, or 190 °C) alone or in combination with pelleting on concentrations of ergot alkaloids. Fifteen samples of ergot-contaminated grain from Alberta and Saskatchewan were assayed for R and S epimers of six alkaloids (ergocryptine, ergocristine, ergocornine, ergometrine, ergosine, and ergotamine) using HPLC MS/MS. Five samples with distinct alkaloid profiles were then selected for heating and pelleting studies. Heating resulted in a linear increase (p < 0.05) of total R and total S epimers with increasing temperature, although some individual R epimers were stable (ergometrine, ergosine, ergotamine). Pelleting also increased (p < 0.05) concentrations of total R and total S epimers detected, although ergometrine concentration decreased (p < 0.05) after pelleting. A feeding study arranged in a 2 × 2 factorial structure used 48 backgrounding Angus-cross steers fed four different diets: (1) Control Mash (CM, no added ergot), (2) Control Pellet (CP), (3) Ergot Mash (EM), or (4) Ergot Pellet (EP). Pelleting heated the ergot to 90–100 °C under 4 bars pressure, but the ergot used in the feeding study was not otherwise heated. Alkaloid concentrations of EM and EP varied by up to 1.1 mg/kg depending on the feed matrix assayed. No differences among treatments were noted for growth performance, feed intake, feed conversion, concentrations of serum prolactin and haptoglobin, hair cortisol, or in temperatures of extremities measured by infrared thermography. The only negative impacts of ergot alkaloids were on blood parameters indicative of reduced immune function or chronic inflammation. Pelleting did not heighten the negative clinical outcomes of ergot, although alkaloid concentrations of pelleted feed increased depending on the matrix assayed. It was hypothesized that the heat and pressure associated with pelleting may enhance the recovery of alkaloids from pelleted feed.

Ammonization of the R- and S-Epimers of Ergot Alkaloids to Assess Detoxification Potential.

Detoxification of ergot-contaminated feed by ammonia would be a practical application, given that ammonia is routinely used in the agriculture industry. To assess the effects of ammonia on ergot alkaloids, natural ergot-contaminated wheat was ammoniated. The total concentration of ergot alkaloids (R- and S-epimers) decreased after exposure to ammonia (8–29%). Separately, the total R-epimers decreased in concentration (40–66%), whereas the total S-epimers increased (21–81%). Specific ergot alkaloids demonstrated degradation and/or epimerization after exposure to ammonia, potentially associated with structural differences, and influenced the total concentrations observed. Ammonization of ergot standards resulted in potential degradation products and epimerization, supporting the above results. The use of ultrahigh-performance liquid chromatography–tandem mass spectrometry provides an updated assessment of the detoxification potential of ammonia for ergot alkaloids and the quantification of the S-epimers. Ammonia alters the R- and S-epimers of ergot alkaloids, which may lead to a potential practical detoxification process of ergot-contaminated feed.

Ergot in Canadian cereals – relevance, occurrence, and current status

Ergot is a fungal disease that occurs on cereals and grasses, including rye, oat, wheat, and barley. The ergot pathogen, Claviceps purpurea, infects the floret, and as infection progresses, it replaces individual kernels, appearing on the head as darkly coloured fungal bodies called sclerotia. Ergot is a grain-grading factor in Canada and can affect grain quality and value, as well as safety due to production of ergot alkaloids. Testing of durum fortified with ergot sclerotia showed that the presence of sclerotia resulted in black specks in semolina and pasta, but had no impact on milling performance or end-product quality. Surveillance performed as part of the Canadian Grain Commission’s annual Harvest Sample and Cargo Monitoring Programs provided insights into ergot incidence and severity in different grains, growing locations, and years, as well as the presence of ergot alkaloids. Analysis of more than 230 000 Harvest Sample Program grain samples from western Canada across 25 years showed the highest ergot incidence and severity occurred in rye, followed by bread and durum wheat, and then barley and oats. Ergot incidence and severity varied annually, but only incidence increased over time. Analyses of bulk grain shipments indicated that the presence of ergot alkaloids reflected the disease incidence observed in the harvest samples (median ergot alkaloid concentrations in wheat and durum > oats and barley). Data on ergot alkaloids from the Cargo Monitoring Program also showed that the Canadian bulk grain-handling system mitigated the variability in annual ergot incidence and severity observed in harvest samples.

Impact of ergot-contaminated feed on yellow mealworm larvae performance and diet preference

This study aimed to determine if ergot alkaloids (EA) would accumulate in yellow mealworm larvae (YML, Tenebrio molitor) when present in their diets and investigate effects on production and survival. Larvae were reared on one of four diets: a control, low, medium, and high containing 63, 3,863, 8,471 and 15,316 μg/kg total EA, respectively. Each diet had five replicates with 150 YML per replicate totalling 3,000 for the 21-day trial. Initial and final weights of the feed and larvae were collected. Ergot alkaloid concentrations in YML at d 21 were 32.6, 94.0 and 155.5 μg/kg in the low, medium, and high treatments respectively, with none detected in those fed the control diet. The frass from YML fed the control, low, medium and high diets contained 18, 364, 1,094, and 1,424 μg/kg total EA, respectively. Feed intake was reduced in larvae fed the low, medium and high treatments relative to the control at 23.3-24.9 g/21 d compared to 30.1 g in the control (P=0.02). Feed-to-gain ratios, average daily gain, and final body weights did not differ among treatments (P&gt;0.05). The larvae did not display any preference for diets when allowed to choose between the four diets (P&gt;0.05). Larvae accumulated only low levels of EA from their diets and although feed intake was depressed, growth was maintained. Further research is required to determine the safety of yellow mealworm reared on EA-contaminated diets.

Evolution of the Ergot Alkaloid Biosynthetic Gene Cluster Results in Divergent Mycotoxin Profiles in Claviceps purpurea Sclerotia.

Research into ergot alkaloid production in major cereal cash crops is crucial for furthering our understanding of the potential toxicological impacts of Claviceps purpurea upon Canadian agriculture and to ensure consumer safety. An untargeted metabolomics approach profiling extracts of C. purpurea sclerotia from four different grain crops separated the C. purpurea strains into two distinct metabolomic classes based on ergot alkaloid content. Variances in C. purpurea alkaloid profiles were correlated to genetic differences within the lpsA gene of the ergot alkaloid biosynthetic gene cluster from previously published genomes and from newly sequenced, long-read genome assemblies of Canadian strains. Based on gene cluster composition and unique polymorphisms, we hypothesize that the alkaloid content of C. purpurea sclerotia is currently undergoing adaptation. The patterns of lpsA gene diversity described in this small subset of Canadian strains provides a remarkable framework for understanding accelerated evolution of ergot alkaloid production in Claviceps purpurea.

Ergot Alkaloid Contents in Hybrid Rye are Reduced by Breeding

Contamination by ergot caused by the phytopathogenic fungus Claviceps purpurea is a constant threat to the whole rye value chain. Ergot alkaloids (EA) produced within the fungal sclerotia are toxic for humans and animals and are subjected to strict regulations in human food. Our main objective was to analyze whether less susceptible rye cultivars with a lower content of sclerotia also contain fewer ergot alkaloids (EA). We analyzed 15 factorial single crosses in multi-environmental trials with artificial inoculation for their ergot severity, the content of twelve EAs by HPLC, and the total ergot content by ELISA. The genotypes displayed a wide range of pollen shedding from fully sterile to fully fertile, of ergot severity expressed as percentage of sclerotia relative to the harvest (0.22–11.47%), and of EA contents when analyzed by HPLC (0.57–45.27 mg/kg. Entry-mean heritabilities were high throughout (0.87–0.98). The factorial analysis yielded a preponderance of male general combining ability (GCA) variances, the estimates for the females were smaller, although significant. EA contents measured by ELISA were, on average, seven times larger. The correlation between ergot severity and EA contents determined by HPLC was r = 0.98 (p ≤ 0.01) and only somewhat lower when analyzed by ELISA. In conclusion, less ergot prone rye genotypes also support lower EA contents.

Cleaving Ergot Alkaloids by Hydrazinolysis-A Promising Approach for a Sum Parameter Screening Method.

Ergot alkaloids are mycotoxins formed by fungi of the Claviceps genus, which are some of the most common contaminants of food and feed worldwide. These toxins are a structurally heterogeneous group of compounds, sharing an ergoline backbone. Six structures and their corresponding stereoisomers are typically quantified by either HPLC-FLD or HPLC-MS/MS and the values subsequently summed up to determine the total ergot alkaloid content. For the development of a screening method targeting all ergot alkaloids simultaneously, the alkaloids need to be transferred to one homogeneous structure: a lysergic acid derivative. In this study, two promising cleaving methods—acidic esterification and hydrazinolysis—are compared, using dihydroergocristine as a model compound. While the acidic esterification proved to be unsuitable, due to long reaction times and oxidation sensitivity, hydrazinolysis reached a quantitative yield in 40‒60 min. Parallel workup of several samples is possible. An increasing effect on the reaction rate by the addition of ammonium iodide was demonstrated. Application of hydrazinolysis to a major ergot alkaloid mix solution showed that all ergopeptines were cleaved, but ergometrine/-inine was barely affected. Still, hydrazinolysis is a suitable tool for the development of a sum parameter screening method for ergot alkaloids in food and feed.

Determining the Rate of Ergot-alkaloid Degradation in Toxic Endophyte-infected Tall Fescue Hay and Haylage During Long Term Storage

Abstract The objectives of this experiment were to determine changes in the nutritive value and ergot alkaloid concentrations of endophyte-infected tall fescue hay and haylage during a 180-d storage period. Forage from a single field of Kentucky-31 tall fescue was cut for hay in late June and allowed to dry in the field. The dry matter (DM) of the windrow of cut forage was measured every 2 h after clipping. Forage was sampled from the windrow in 6 location blocks once forage DM reached target levels for haylage and hay treatments. Haylage and hay samples were taken when the DM of the windrow reached 50% and 80%, respectively. Seven subsamples of each treatment within block were chopped to 1.91 cm in length with a lettuce chopper and vacuum sealed in oxygen-excluding bags. Sample bags were stored indoors and opened at 30 d intervals over the 180-d storage period. Samples were analyzed for pH, nutritive value, and individual ergot alkaloid concentrations using high-performance liquid chromatography. Within each storage day, treatment within block was considered the experimental unit. Data were analyzed in SAS using the MIXED procedure with fixed effects of treatment, day, and the treatment by day interaction. Location block was considered a random effect. As expected, pH was decreased for haylage compared to hay at all time points (P &amp;lt; 0.01) and DM was greater (P &amp;lt; 0.01) for hay compared to haylage. Neutral detergent fiber values were greater (P &amp;lt; 0.01) for hay compared to haylage and declined during storage (P &amp;lt; 0.01). Total ergot alkaloid concentrations did not differ by treatment (P = 0.61), but ergovaline concentrations declined (P &amp;lt; 0.01) during storage. Collectively, these results indicate minimal differences in nutritive value and ergot alkaloid concentrations between hay and haylage during storage, and that ergovaline concentrations decline during storage.

Occurrence of Ergot Alkaloids in Barley and Wheat from Algeria.

The natural occurrence of six major ergot alkaloids, ergometrine, ergosine, ergotamine, ergocornine, ergokryptine and ergocristine, as well as their corresponding epimers, were investigated in 60 cereal samples (barley and wheat) from Algeria. Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) and a QuEChERS extraction method were used for sample analysis. The results revealed that 12 out of 60 samples (20%) were contaminated with ergot alkaloids. Wheat was the most contaminated matrix, with an incidence of 26.7% (8 out of 30 samples). The concentration of total ergot alkaloids ranged from 17.8 to 53.9 µg/kg for barley and from 3.66 to 76.0 μg/kg for wheat samples. Ergosine, ergokryptine and ergocristine showed the highest incidences in wheat, while ergometrine was the most common ergot in barley.

Vasoactive Effects of Acute Ergot Exposure in Sheep

Ergotism is a common and increasing problem in Saskatchewan’s livestock. Chronic exposure to low concentrations of ergot alkaloids is known to cause severe arterial vasoconstriction and gangrene through the activation of adrenergic and serotonergic receptors on vascular smooth muscles. The acute vascular effects of a single oral dose with high-level exposure to ergot alkaloids remain unknown and are examined in this study. This study had two main objectives; the first was to evaluate the role of α1-adrenergic receptors in mediating the acute vasocontractile response after single-dose exposure in sheep. The second was to examine whether terazosin (TE) could abolish the vascular contractile effects of ergot alkaloids. Twelve adult female sheep were randomly placed into control and exposure groups (n = 6/group). Ergot sclerotia were collected and finely ground. The concentrations of six ergot alkaloids (ergocornine, ergocristine, ergocryptine, ergometrine, ergosine, and ergotamine) were determined using HPLC/MS at Prairie Diagnostic Services Inc., (Saskatoon, SK, Canada). Each ewe within the treatment group received a single oral treatment of ground ergot sclerotia at a dose of 600 µg/kg BW (total ergot) while each ewe in the control group received water. Animals were euthanized 12 h after the treatment, and the pedal artery (dorsal metatarsal III artery) from the left hind limb from each animal was carefully dissected and mounted in an isolated tissue bath. The vascular contractile response to phenylephrine (PE) (α1-adrenergic agonist) was compared between the two groups before and after TE (α1-adrenergic antagonist) treatment. Acute exposure to ergot alkaloids resulted in a 38% increase in vascular sensitivity to PE compared to control (Ctl EC50 = 1.74 × 10−6 M; Exp EC50 = 1.079 × 10−6 M, p = 0.046). TE treatment resulted in a significant dose-dependent increase in EC50 in both exposure and control groups (p < 0.05 for all treatments). Surprisingly, TE effect was significantly more pronounced in the ergot exposed group compared to the control group at two of the three concentrations of TE (TE 30 nM, p = 0.36; TE 100 nM, p < 0.001; TE 300 nM, p < 0.001). Similar to chronic exposure, acute exposure to ergot alkaloids results in increased vascular sensitivity to PE. TE is a more potent dose-dependent antagonist for the PE contractile response in sheep exposed to ergot compared to the control group. This study may indicate that the dry gangrene seen in sheep, and likely other species, might be related to the activation of α1-adrenergic receptor. This effect may be reversed using TE, especially at early stages of the disease before cell death occurs. This study may also indicate that acute-single dose exposure scenario may be useful in the study of vascular effects of ergot alkaloids.

Determination of principal ergot alkaloids in swine feeding.

Ergot alkaloids are secondary metabolites produced by fungi in the genus Claviceps. They contaminate a large variety of cereals, such as rye, triticale, wheat and barley. The ingestion of contaminated cereals might cause adverse health effects in humans and animals. In fact, pigs, cattle, sheep, and poultry are involved in sporadic outbreaks and, although there are several studies about occurrence of ergot alkaloids in grain cereals, there are scarce studies focused on compound feed. Twelve ergot alkaloids have been quantified in 228 feed samples intended for swine. The analytes were extracted using QuEChERS with Z-Sep+ as sorbent in the clean-up step, which reduced the matrix effect, allowing limits of quantification between 2.1 and 21.7μg kg-1 . The analytes were subsequently quantified by ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS). A total of 29 samples (12.7%) revealed contamination by at least one ergot alkaloid, and among contaminated samples, 65% were contaminated by more than one. Only 6 of 12 target ergot alkaloids showed concentrations above the limit of quantification. The concentrations for individual ergot alkaloids ranged between 5.9μg kg-1 for ergosinine to 145.3μg kg-1 for ergometrine (the predominant ergot alkaloid), while the total ergot alkaloid content ranged from 5.9 to 158.7μg kg-1 . The occurrence of ergot alkaloids in feed samples in Spain seems to be lower than in other regions of Europe. All the samples fulfilled current recommendations of the feed industry about practical limits for ergot alkaloids in pig feeds. This suggests that the feeds are safe for pig consumption, regarding the presence of ergot alkaloids. © 2021 Society of Chemical Industry.