Detection of Endophyte Mycotoxins by Service Laboratories: Providing Answers for Safe Feed

. A global network of service laboratories exists to test livestock feed materials (typically grass hay and pellets) for ergovaline, ergot alkaloids and lolitrem B to ensure ‘safe feeds’ are being given to livestock. These compounds are mycotoxins produced by endophytic fungi that naturally reside in feed material. They have been purposely bred into grass species, as they enhance the plant’s survival from drought and insect predation. Unfortunately, ergovaline and other ergot alkaloids also cause vasoconstrictive effects and reproductive difficulties in livestock, resulting in a $1 million annual loss in production for the cattle industry in the USA alone. Lolitrem B is a neurotoxicant that causes a syndrome known as ‘ryegrass staggers,’ which involves a tremoring response in the animal. Clients of these service laboratories include hay farmers who want to be confident that the product they are selling is safe to feed to livestock; veterinarians who are trying to rule out causes of disease in clinical cases; individuals wanting to check personal feed sources; and researchers investigating innovative solutions to these feed contaminants. Each year, approximately 33,000 containers of hay are shipped overseas from the USA, including Pacific Rim and Middle Eastern countries, bringing in an estimated$130 million annually. If the importing country requires it, the material in these containers must be tested for the appropriate mycotoxin(s) and have a certificate stating that the level found was below the established threshold of toxicity. Discussion of sample submission, analysis and result receipt will be compared amongst international laboratories known to perform analyses for these mycotoxins

Urinary excretion of the mycotoxins lolitrem B and lysergic acid in cattle consuming perennial ryegrass straw over 56 days

Perennial ryegrass (Lolium perenne) hay is an important source of forage for a variety of livestock species, and is most common in cool, coastal climates such as the Pacific Northwest. An endophyte fungus called Neotyphodium lolii frequently colonizes this grass to promote insect resistance, drought tolerance, and growth enhancement without the need to incorperate excessive use of fertilizers, irrigation or pesticides. Unfortunately, encouragement of endophyte infection can also result in the production of mycotoxins including lolitrem B and ergot alkaloids. Lolitrem B is a tremorgenic neurotoxin that effects livestock and other herbivores and is responsible for the condition known as “ryegrass staggers,” in which animals develop tremors, ataxia and frequently collapse. The ergot alkaloids are vasoconstrictors and prolactin inhibitors that negatively affect thermoregulation, reproductive efficiency and milk yield. The toxicokinetics of lolitrem B have not yet been fully elucidated; however, that of the ergot alkaloid mycotoxin ergovaline has, and the main urinary metabolic breakdown product was determined to be lysergic acid. A thorough understanding of the metabolism of lolitrem B/ergot alkaloids from perennial ryegrass in cattle must be established before a risk assessment can be extended to humans. Therefore, the goal of this project was to evaluate one toxicokinetic compartment, the urine, of cattle fed varying doses of lolitrem B- and ergovaline-containing perennial ryegrass hay for 60 days for lolitrem B and the ergot alkaloid breakdown product lysergic acid via LC-MS/MS. Our experiment confirmed that lolitrem B is not secreted in bovine urine; lysergic was detected in a dose-dependent manner. The tendency for lysergic acid to be excreted in aqueous matrices raises concerns for human consumption of milk products from exposed animals

Metagenomic Insights into the RDX-Degrading Potential of the Ovine Rumen Microbiome

The manufacturing processes of royal demolition explosive (RDX), or hexahydro-1,3,5-trinitro-1,3,5-triazine, have resulted in serious water contamination. As a potential carcinogen, RDX can cause a broad range of harmful effects to humans and animals. The ovine rumen is capable of rapid degradation of nitroaromatic compounds, including RDX. While ruminal RDX-degrading bacteria have been identified, the genes and pathways responsible for RDX degradation in the rumen have yet to be characterized. In this study, we characterized the metabolic potential of the ovine rumen using metagenomic approaches. Sequences homologous to at least five RDX-degrading genes cloned from environmental samples (diaA, xenA, xenB, xplA, and xplB) were present in the ovine rumen microbiome. Among them, diaA was the most abundant, likely reflective of the predominance of the genus Clostridium in the ovine rumen. At least ten genera known to harbor RDX-degrading microorganisms were detectable. Metagenomic sequences were also annotated using public databases, such as Pfam, COG, and KEGG. Five of the six Pfam protein families known to be responsible for RDX degradation in environmental samples were identified in the ovine rumen. However, increased substrate availability did not appear to enhance the proliferation of RDX-degrading bacteria and alter the microbial composition of the ovine rumen. This implies that the RDX-degrading capacity of the ovine rumen microbiome is likely regulated at the transcription level. Our results provide metagenomic insights into the RDX-degrading potential of the ovine rumen, and they will facilitate the development of novel and economic bioremediation strategies

Survival and development of potato psyllid (Hemiptera: Triozidae) on Convolvulaceae: Effects of a plant-fungus symbiosis (Periglandula)

Plant species in the family Solanaceae are the usual hosts of potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Psylloidea: Triozidae). However, the psyllid has also been shown to develop on some species of Convolvulaceae (bindweeds and morning glories). Developmental success on Convolvulaceae is surprising given the rarity of psyllid species worldwide associated with this plant family. We assayed 14 species of Convolvulaceae across four genera (Convolvulus, Calystegia, Ipomoea, Turbina) to identify species that allow development of potato psyllid. Two populations of psyllids were assayed (Texas, Washington). The Texas population overlaps extensively with native Convolvulaceae, whereas Washington State is noticeably lacking in Convolvulaceae. Results of assays were overlain on a phylogenetic analysis of plant species to examine whether Convolvulaceae distantly related to the typical host (potato) were less likely to allow development than species of Convolvulaceae more closely related. Survival was independent of psyllid population and location of the plant species on our phylogenetic tree. We then examined whether presence of a fungal symbiont of Convolvulaceae (Periglandula spp.) affected psyllid survival. These fungi associate with Convolvulaceae and produce a class of mycotoxins (ergot alkaloids) that may confer protection against plant-feeding arthropods. Periglandula was found in 11 of our 14 species, including in two genera (Convolvulus, Calystegia) not previously known to host the symbiont. Of these 11 species, leaf tissues from five contained large quantities of two classes of ergot alkaloids (clavines, amides of lysergic acid) when evaluated by LC-MS/MS. All five species also harbored Periglandula. No ergot alkaloids were detected in species free of the fungal symbiont. Potato psyllid rapidly died on the five species that harbored Periglandula and contained ergot alkaloids, but survived to adulthood on seven of the nine species in which ergot alkaloids were not detected. These results support the hypothesis that a plant fungus symbiotic relationship affects the suitability of certain Convolvulaceae to potato psyllid

Cases of ergotism in livestock and associated ergot alkaloid concentrations in feed

Ergot-induced disease was known long before Biblical times and has been the root cause for countless human epidemics spanning from the early fourteenth century to the late sixteenth century. In contrast, many of these same ergot alkaloids have been utilized for their medicinal properties to mitigate migraine headaches and have had indications as anticarcinogens. Although ergot alkaloids have been used for centuries, basic pharmacokinetic data has not been documented for clinical disease. Consequently, a threshold dose and accurate dose-response data have yet to be established. Throughout the past several years, new detection techniques have emerged to detect these alkaloids at the parts per billion which have allowed for new efforts to be made with respect to determining threshold levels and making accurate clinical diagnoses. This perspectives article provides a critical initial step for establishing a uniform interpretation of ergot toxicosis from limited existing data

Persia URSA Lolitrem B Final.pptx

Perennial ryegrass (Lolium perenne) hay is an important source of forage for a variety of livestock species, and is most common in cool, coastal climates such as the Pacific Northwest. An endophyte fungus called Neotyphodium lolii frequently colonizes this grass to promote insect resistance, drought tolerance, and growth enhancement without the need to incorperate excessive use of fertilizers, irrigation or pesticides. Unfortunately, encouragement of endophyte infection can also result in the production of mycotoxins including lolitrem B and ergot alkaloids. Lolitrem B is a tremorgenic neurotoxin that effects livestock and other herbivores and is responsible for the condition known as “ryegrass staggers,” in which animals develop tremors, ataxia and frequently collapse. The ergot alkaloids are vasoconstrictors and prolactin inhibitors that negatively affect thermoregulation, reproductive efficiency and milk yield. The toxicokinetics of lolitrem B have not yet been fully elucidated; however, that of the ergot alkaloid mycotoxin ergovaline has, and the main urinary metabolic breakdown product was determined to be lysergic acid. A thorough understanding of the metabolism of lolitrem B/ergot alkaloids from perennial ryegrass in cattle must be established before a risk assessment can be extended to humans. Therefore, the goal of this project was to evaluate one toxicokinetic compartment, the urine, of cattle fed varying doses of lolitrem B- and ergovaline-containing perennial ryegrass hay for 60 days for lolitrem B and the ergot alkaloid breakdown product lysergic acid via LC-MS/MS. Our experiment confirmed that lolitrem B is not secreted in bovine urine; lysergic was detected in a dose-dependent manner. The tendency for lysergic acid to be excreted in aqueous matrices raises concerns for human consumption of milk products from exposed animals

Composition and antibacterial activity of essential oils obtained from plants of the Lamiaceae family against pathogenic and beneficial bacteria

The qualitative composition and antibacterial activity of six essential oils obtained from plants cultivated in the Colombian Andes (Mentha spicata, Mentha piperita, Ocimum basilicum, Salvia officinalis, Rosmarinus officinalis and Thymus vulgaris) and a commercial essential oil of Origanum vulgare subsp. hirtum were investigated. The essential oil composition was determined by gas chromatography-mass spectrometry (GC-MS), while the antibacterial activity of the essential oils against Escherichia coli, Salmonella enteritidis, Salmonella typhimurium, Lactobacillus acidophilus and Bifidobacterium breve was measured as the minimum bacte icidal concentration (MBC) using the agar dilution method. The chemical analysis revealed the presence of 16-28 compounds in each oil, corresponding mainly to phenols, oxygenated and hydrocarbon monoterpenes. O. vulgare and T. vulgaris oils were active at low MBCs (MBC . 5 mg/ml) against all bacteria evaluated, including beneficial microorganisms. In contrast, O. basilicum oil was more active against pathogenic bacteria (MBCs . 10mg/ml) than beneficial bacteria (MBCs of 80 mg/ml). The present study shows that the antimicrobial potential of essential oils depends not only on the chemical composition of the oil but also on the targeted microorganism. This has important practical implications for essential oils intended to be used as feed additives with antibacterial properties for animal nutrition or pharmaceutical products with natural compounds.Pesquisou-se a composicao qualitativo e a atividade antibacteriana de seis azeites essenciais obtidos de plantas cultivadas nos Andes Colombianos (Mentha spicata, Mentha piperita, Ocimum basilicum, Salvia officinalis, Rosmarinus officinalis e Thymus vulgaris) e um azeite essencial comercial de Origanum vulgare subsp. hirtum. A composicao dos azeites essenciais foi determinada por cromatografia de gases - espectrofotometria de massas (CM-EM), enquanto a atividade antibacteriana dos azeites essenciais contra Escherichia coli, Salmonella enteritidis, Salmonella typhimurim, Lactobacillus acidophilus e Bifidobacterium breve foi medida como a concentracao minima bactericida (CMB) usando o metodo de diluicao em agar. As analises quimicas revelaram a presenca de16 . 28 compostos em cada azeite, correspondendo principalmente a monoterpenos fenolicos, hidrocarbonetos e oxigenados. Os azeites de O. vulgare e T. vulgaris foram ativos contra todas as bacterias testadas, incluindo microorganismos beneficos a CMBs baixas (CMB . 5 mg/ml). Em contraste, o azeite de O. basilicum foi mais ativo contra bacterias patogenicas do que bacterias beneficas (CMBs de 80 mg/ml). Este estudo demonstrou o potencial antimicrobiano dos azeites essenciais depende da composicao quimica do azeite e o microorganismo proprio. Estes resultados tem implicacoes praticas para os azeites essenciais usados como aditivos alimenticios com propriedades antibacterianas para a nutricao animal ou produtos farmaceuticos com produtos naturais.Se investigo la composicion cualitativa y la actividad antibacteriana de seis aceites esenciales obtenidos de plantas cultivadas en los Andes Colombianos (Mentha spicata, Mentha piperita, Ocimum basilicum, Salvia officinalis, Rosmarinus officinalis y Thymus vulgaris) y un aceite esencial comercial de Origanum vulgare subsp. hirtum. La composicion de los aceites esenciales fue determinada por cromatografia de gases- espectrofotometria de masas (CG-EM), mientras que la actividad antibacteriana de los aceites esenciales contra Escherichia coli, Salmonella enteritidis, Salmonella typhimurim, Lactobacillus acidophilus y Bifidobacterium breve, fue medida como la concentracion minima bactericida (CMB) usando el metodo de dilucion en agar. Los analisis quimicos revelaron la presencia de16 . 28 compuestos en cada aceite, correspondiendo principalmente a monoterpenos fenolicos, oxigenados e hidrocarbonos. Los aceites de O. vulgare y T. vulgaris fueron activos contra todas las bacterias evaluadas, incluyendo microorganismos beneficos a CMBs bajas (CMB . 5 mg/ml). En contraste, el aceite de O. basilicum fue mas activo contra bacterias patogenas (CMBs . 10 mg/ml) en comparacion de bacterias beneficas (CMBs de 80 mg/ml). El presente estudio demostro que el potencial antimicrobiano de los aceites esenciales no depende solo de la composicion quimica del aceite sino tambien del microorganismo por si mismo. Estos resultados tienen implicaciones practicas para los aceites esenciales usados como aditivos alimenticios con propiedades antibacterianas para la nutricion animal o productos farmaceuticos con compuestos naturales

Metagenomic insights into the RDX-degrading potential of the ovine rumen microbiome.

The manufacturing processes of royal demolition explosive (RDX), or hexahydro-1,3,5-trinitro-1,3,5-triazine, have resulted in serious water contamination. As a potential carcinogen, RDX can cause a broad range of harmful effects to humans and animals. The ovine rumen is capable of rapid degradation of nitroaromatic compounds, including RDX. While ruminal RDX-degrading bacteria have been identified, the genes and pathways responsible for RDX degradation in the rumen have yet to be characterized. In this study, we characterized the metabolic potential of the ovine rumen using metagenomic approaches. Sequences homologous to at least five RDX-degrading genes cloned from environmental samples (diaA, xenA, xenB, xplA, and xplB) were present in the ovine rumen microbiome. Among them, diaA was the most abundant, likely reflective of the predominance of the genus Clostridium in the ovine rumen. At least ten genera known to harbor RDX-degrading microorganisms were detectable. Metagenomic sequences were also annotated using public databases, such as Pfam, COG, and KEGG. Five of the six Pfam protein families known to be responsible for RDX degradation in environmental samples were identified in the ovine rumen. However, increased substrate availability did not appear to enhance the proliferation of RDX-degrading bacteria and alter the microbial composition of the ovine rumen. This implies that the RDX-degrading capacity of the ovine rumen microbiome is likely regulated at the transcription level. Our results provide metagenomic insights into the RDX-degrading potential of the ovine rumen, and they will facilitate the development of novel and economic bioremediation strategies

GiarrizzoJuanVetMedMetagenomicInsightsRDX.pdf

The manufacturing processes of royal demolition explosive (RDX), or hexahydro-1,3,5-trinitro-1,3,5-triazine, have resulted in serious water contamination. As a potential carcinogen, RDX can cause a broad range of harmful effects to humans and animals. The ovine rumen is capable of rapid degradation of nitroaromatic compounds, including RDX. While ruminal RDX-degrading bacteria have been identified, the genes and pathways responsible for RDX degradation in the rumen have yet to be characterized. In this study, we characterized the metabolic potential of the ovine rumen using metagenomic approaches. Sequences homologous to at least five RDX-degrading genes cloned from environmental samples (diaA, xenA, xenB, xplA, and xplB) were present in the ovine rumen microbiome. Among them, diaA was the most abundant, likely reflective of the predominance of the genus Clostridium in the ovine rumen. At least ten genera known to harbor RDX-degrading microorganisms were detectable. Metagenomic sequences were also annotated using public databases, such as Pfam, COG, and KEGG. Five of the six Pfam protein families known to be responsible for RDX degradation in environmental samples were identified in the ovine rumen. However, increased substrate availability did not appear to enhance the proliferation of RDX-degrading bacteria and alter the microbial composition of the ovine rumen. This implies that the RDX-degrading capacity of the ovine rumen microbiome is likely regulated at the transcription level. Our results provide metagenomic insights into the RDX-degrading potential of the ovine rumen, and they will facilitate the development of novel and economic bioremediation strategies

GiarrizzoJuanVetMedMetagenomicInsightsRDX_SupportingInformation.zip

The manufacturing processes of royal demolition explosive (RDX), or hexahydro-1,3,5-trinitro-1,3,5-triazine, have resulted in serious water contamination. As a potential carcinogen, RDX can cause a broad range of harmful effects to humans and animals. The ovine rumen is capable of rapid degradation of nitroaromatic compounds, including RDX. While ruminal RDX-degrading bacteria have been identified, the genes and pathways responsible for RDX degradation in the rumen have yet to be characterized. In this study, we characterized the metabolic potential of the ovine rumen using metagenomic approaches. Sequences homologous to at least five RDX-degrading genes cloned from environmental samples (diaA, xenA, xenB, xplA, and xplB) were present in the ovine rumen microbiome. Among them, diaA was the most abundant, likely reflective of the predominance of the genus Clostridium in the ovine rumen. At least ten genera known to harbor RDX-degrading microorganisms were detectable. Metagenomic sequences were also annotated using public databases, such as Pfam, COG, and KEGG. Five of the six Pfam protein families known to be responsible for RDX degradation in environmental samples were identified in the ovine rumen. However, increased substrate availability did not appear to enhance the proliferation of RDX-degrading bacteria and alter the microbial composition of the ovine rumen. This implies that the RDX-degrading capacity of the ovine rumen microbiome is likely regulated at the transcription level. Our results provide metagenomic insights into the RDX-degrading potential of the ovine rumen, and they will facilitate the development of novel and economic bioremediation strategies