The Bioactive Value of Tamarix gallica Honey from Different Geographical Origins.

This study was conducted to assess the bioactive value of Tamarix gallica honey samples collected from three countries. In total, 150 Tamarix gallica honey samples from Saudi Arabia (50), Libya (50), and Egypt (50) were collected and compared, based on the results of the melissopalynological analysis, their physicochemical attributes, antioxidant and antimicrobial activities, and biochemical properties, together with their total phenolic and total flavonoid contents. Depending on the geographical origin, we observed different levels of growth suppression for six resistant bacterial strains. The pathogenic microorganisms tested in this study were Staphylococcus aureus, Streptococcus mutans, Klebsiella pneumoniae, Escherichia coli, Proteus vulgaris, and Pseudomonas aeruginosa. There was a strong correlation between the polyphenol and flavonoid contents, as well as significant (p < 0.05) radical scavenging activities. The melissopalynological analysis and physicochemical properties complied with the recommendation of the Gulf and Egyptian Technical Regulations on honey, as well as the Codex Alimentarius of the World Health Organization and the European Union Normative related to honey quality. It was concluded that Tamarix gallica honey from the three countries has the capacity to suppress pathogenic bacterial growth and has significant radical scavenging activities. Moreover, these findings suggest that Tamarix gallica honey may be considered as an interesting source of antimicrobial compounds and antioxidants for therapeutical and nutraceutical industries or for food manufacturers.

Case-Control Study of Nodding Syndrome in Acholiland: Urinary Multi-Mycotoxin Screening.

This case-control study adds to the growing body of knowledge on the medical, nutritional, and environmental factors associated with Nodding Syndrome (NS), a seizure disorder of children and adolescents in northern Uganda. Past research described a significant association between NS and prior history of measles infection, dependence on emergency food and, at head nodding onset, subsistence on moldy maize, which has the potential to harbor mycotoxins. We used LC-MS/MS to screen for current mycotoxin loads by evaluating nine analytes in urine samples from age-and-gender matched NS cases (n = 50) and Community Controls (CC, n = 50). The presence of the three mycotoxins identified in the screening was not significantly different between the two groups, so samples were combined to generate an overall view of exposure in this community during the study. Compared against subsequently run standards, α-zearalenol (43 ± 103 µg/L in 15 samples > limit of quantitation (LOQ); 0 (0/359) µg/L), T-2 toxin (39 ± 81 µg/L in 72 samples > LOQ; 0 (0/425) µg/L) and aflatoxin M1 (4 ± 10 µg/L in 15 samples > LOQ; 0 (0/45) µg/L) were detected and calculated as the average concentration ± SD; median (min/max). Ninety-five percent of the samples had at least one urinary mycotoxin; 87% were positive for two of the three compounds detected. While mycotoxin loads at NS onset years ago are and will remain unknown, this study showed that children with and without NS currently harbor foodborne mycotoxins, including those associated with maize.

Molecular and Alkaloid Characterization of Claviceps purpurea Sensu Lato From Grass Seed Production Areas of the U.S. Pacific Northwest.

Ergot, caused by Claviceps purpurea sensu lato, is an economically important seed replacement disease of Kentucky bluegrass (Poa pratensis) and perennial ryegrass (Lolium perenne) seed crops. C. purpurea sensu stricto is considered the primary Claviceps species responsible, but genetic diversity and cryptic species within C. purpurea sensu lato have previously been reported. Fifty-six C. purpurea sensu lato isolates collected from P. pratensis (n = 21) and L. perenne (n = 35) in Oregon and Washington between 2010 and 2014 were characterized via random amplified polymorphic DNA (RAPD), partial internal transcribed spacer (ITS), ß-tubulin and elongation factor-1α (EF-1α) sequences, conidial size, and ergot alkaloid chemotype. Based on RAPD analysis, seven isolates from P. pratensis and 33 isolates from L. perenne collected in Oregon corresponded to C. purpurea sensu stricto, and 13 isolates collected from P. pratensis in Washington and Oregon were identified as C. humidiphila. Partial ITS, ß-tubulin, and EF-1α sequences identified 10 isolates from P. pratensis as C. humidiphila, and seven isolates from P. pratensis and 33 isolates from L. perenne were identified as C. purpurea sensu stricto. Several isolates generated ambiguous RAPD bands or sequences that prevented identification. Ergot alkaloid chemotype profiling found that ergocornine and its epimer were predominant in sclerotia from P. pratensis, whereas ergotamine and its epimer were most abundant in sclerotia from L. perenne. This study confirms the presence of the C. purpurea sensu lato species complex in the U.S. Pacific Northwest and suggests that more research is needed to characterize and mitigate Claviceps spp. infection of grass seed crops in North America.

Co-exposure of the Mycotoxins Lolitrem B and Ergovaline in Steers Fed Perennial Ryegrass ( Lolium perenne) Straw: Metabolic Characterization of Excreta.

Past research showed a strong linear correlation between levels of the mycotoxins lolitrem B (LB, a tremorgen) and ergovaline (EV, an ergot alkaloid and potent vasoconstrictor) in perennial ryegrass (PRG) forage. The purpose of this study was to characterize the excretion of these two compounds in beef cattle consuming PRG straw and to utilize liquid chromatography-tandem mass spectrometry to investigate the metabolism of LB and EV in excreta. Four groups of steers ( n = 6/group) were fed endophyte-infected PRG for 64 days (2256/638, 1554/373, 1012/259, or 247/<100 µg/kg LB/EV). Concentrations of LB and EV in both PRG straw and feces showed a linear relationship to each other. Feces reflected a dose-response for both mycotoxins, with values increasing most rapidly through 21 days then plateauing. Urine contained no detectable level of either compound or the ergoline lysergic acid. Screening for metabolites showed oxidation and reduction biotransformations for both toxins, with additional conjugation products detected for ergovaline.

Determination of the Ergot Alkaloid Ergovaline in Tall Fescue Seed and Straw Using a QuEChERS Extraction Method with High-Performance Liquid Chromatography-Fluorescence Detection.

Ergovaline is an ergot alkaloid produced by the symbiotic endophyte Epichloë coenophiala, which can colonize varieties of the cool-season grass tall fescue (Festuca arundinacea). It is the principle toxicant responsible for the vasoconstrictive and reproductive sequelae seen in "fescue toxicosis" in livestock which consume forage exceeding the threshold of toxicity established for this compound. A new method for extraction of ergovaline from tall fescue seed and straw was optimized and validated, on the basis of the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method, with high-performance liquid chromatography-fluorescence detection. Fourteen extraction solvents were tested; 2.1 mM ammonium carbonate/acetonitrile (50/50, v/v) had the highest and most consistent recovery (91-101%). Linearity, limit of detection, limit of quantitation, accuracy,and intra- and interday precisions for tall fescue seed and straw were 100-3500 µg/kg, 37 and 30 µg/kg, 100 µg/kg, 98%, 3.0 and 1.6%, and 3.8 and 1.0%, respectively. When the currently used solid-phase extraction (SPE) and QuEChERS methods were applied to 17 tall fescue straw samples, there was good agreement (correlation coefficient 0.9978). The QuEChERS method achieved the goals of eliminating chlorinated solvents and developing a fast, efficient, reliable method for quantitating ergovaline in tall fescue forage that can be applied in a high-throughput food safety laboratory.

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

Ergot-induced disease in humans 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 anti-carcinogens. Although ergot alkaloids have been used for centuries by humans, basic pharmacokinetic data has not been documented for clinical disease in livestock. 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 (ppb) level which has allowed for new efforts to be made with respect to determining threshold levels and making accurate clinical diagnoses in affected animals. This perspectives article provides a critical initial step for establishing a uniform interpretation of ergot toxicosis from limited existing data.

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.

The role of the Oregon State University Endophyte Service Laboratory in diagnosing clinical cases of endophyte toxicoses.

The Oregon State University Colleges of Veterinary Medicine and Agricultural Sciences instituted the Endophyte Service Laboratory to aid in diagnosing toxicity problems associated with cool-season grasses in livestock. The endophyte (Neotyphodium coenophalum) present in tall fescue (Festuca arundinacea) produces ergopeptine alkaloids, of which ergovaline is the molecule used to determine exposure and toxicity thresholds for the vasoconstrictive conditions "fescue foot" and "summer slump". Another vasoconstrictive syndrome, "ergotism," is caused by a parasitic fungus, Claviceps purpurea, and its primary toxin, ergotamine. "Ryegrass staggers" is a neurological condition that affects livestock consuming endophyte (Neotyphodium lolii)-infected perennial ryegrass (Lolium perenne) with high levels of lolitrem B. HPLC-fluorescent analytical methods for these mycotoxins are described and were used to determine threshold levels of toxicity for ergovaline and lolitrem B in cattle, sheep, horses, and camels. In addition, six clinical cases in cattle are presented to illustrate diagnosis of these three diseases.

Ruminal bioremediation of the high energy melting explosive (HMX) by sheep microorganisms.

The ability of ruminal microorganisms to degrade octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (high melting explosive, HMX) as consortia from whole rumen fluid (WRF), and individually as 23 commercially available ruminal strains, was compared under anaerobic conditions. Compound degradation was monitored by high-performance liquid chromatography, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for delineation of the metabolic pathway. In WRF, 30 µM HMX was degraded to 5 µM HMX within 24 h. Metabolites consistent with m/z 149, 193 and 229 were present throughout the incubation period. We propose that peaks with an m/z of 149 and 193 are arrived at through reduction of HMX to nitroso or hydroxylamino intermediates, then direct enzymatic ring cleavage to produce these HMX derivatives. Possible structures of m/z 229 are still being investigated and require further LC-MS/MS analysis. None of the 23 ruminal strains tested were able to degrade HMX as a pure culture when grown in either a low carbon or low nitrogen basal medium over 120 h. We conclude that microorganisms from the rumen, while sometimes capable as individuals in the bioremediation of other explosives, excel as a community in the case of HMX breakdown.

Biochemical and microbial analysis of ovine rumen fluid incubated with 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX).

In this study, the rumen was assessed for its potential to detoxify RDX using molecular microbial ecology as well as analytical chemistry techniques. Results indicated significant loss (P < 0.05) of RDX in <8-h post incubation, and qualitative LC-MS/MS analysis showed evidence for the formation of 1-NO-RDX (M-O + HCOO) and methylenedinitramine metabolites. A total of 1106 16S rRNA-V3 clones were sequenced, and most sequences associated with either the phyla Bacteroidetes or Firmicutes. A LibCompare analysis for the RDX treatment showed an enrichment (P < 0.01) of the genus Prevotella. From these results, it can be concluded that the rumen is capable of detoxifying RDX, and the members of the genus Prevotella are linked to this detoxification.

ESI-Mass spectrometric and HPLC elucidation of a new ergot alkaloid from perennial ryegrass hay silage associated with bovine reproductive problems.

This case report involves four dairies in the Willamette Valley, Oregon, which experienced reproductive problems associated with the presence of a large, previously unidentified, peak eluting at 5 min in a standard ergovaline high-performance liquid chromatography assay of perennial ryegrass silage fed to those animals. Mycotoxin analysis of the silage was negative, as was serological screening of the herds for infectious bovine rhinotracheitis, bovine diarrhea virus and Leptospirosis, including culturing of urine for Leptospira hardjo hardjobovis. Prolactin concentrations were low in most cattle, consistent with ingestion of ergot alkaloids. We believe that this peak represents a novel ergot alkaloid-related compound due to its extractability with Ergosil, its detectability due to fluorescence, and its chromatographic retention between ergovaline (mw = 533) and ergotamine (mw = 581). Its molecular weight was calculated as 570 owing to the predominance of a m/z 593.5 ion in the full scan ESI(+)MS and its deduced tendency to complex with Na(+) (as m/z 593) or K(+) (as m/z 609) ions. We offer rationales for elucidation of the structure of this compound, with the closest starting point comprising an m.w. of 566-a fructofuranosyl-(2-1)-O-beta-D-fructofuranoside derivative of 6,7-secoergoline from Claviceps fusiformis. This m.w. requires modifications, such as reduction of two double bonds in the secoergoline component to give the target 570 m.w. Despite the lack of a definitive structure, the analysis herein provides a starting point for eventual elucidation of this apparently new ergot alkaloid, and to guide and encourage further investigation as to its association with endophyte toxicosis in livestock.

Design and in vitro evaluation of new rpoB-DGGE primers for ruminants.

Two new primer sets based on the rpoB gene were designed and evaluated with bovine and ovine rumen samples. The newly developed rpoB-DGGE primer set was used along with the 16S rRNA gene-V3, and another (old) rpoB-DGGE-based primer set from a previous study to in vitro compare the bovine and ovine rumen ecosystems. The results indicate a significant (P<0.001) difference in the microbial population between the two ruminants irrespective of the primers used in the analysis. Qualitative comparison of the data provides evidence for the presence of similar phyla profiles between the 16S rRNA gene and the newly developed rpoB primers. A comparison between the two rpoB-based primer sets (old and new) showed that the old rpoB-based primers failed to amplify phylum Bacteroidetes (a common phylum in the rumen) in both bovine and ovine rumen samples. The old and new rpoB-DGGE-based primers amplified a large number of clones belonging to phylum Proteobacteria, providing a useful insight into the microbial structure of the rumen. ChaoI, ACE, Simpson, and Shannon-Weaver index analysis estimated the bovine rumen to be more diverse than the ovine rumen for all three primer sets. These results provide a new insight into the community structure among ruminants using the newly developed primers in this study.

Changes to the rumen bacterial population of sheep with the addition of 2,4,6-trinitrotoluene to their diet.

Previous work has shown that bacterial isolates from the sheep rumen are capable of detoxifying 2,4,6-trinitrotoluene (TNT) into polar constituents. In this study, the dietary effects of TNT on the sheep rumen microbial community were evaluated using molecular microbiology ecology tools. Rumen samples were collected from sheep fed with and without TNT added to their diet, genomic DNA was extracted, and the 16S rRNA-V3 gene marker was used to quantify changes in the microbial population in the rumen. Control and treatment samples yielded 533 sequences. Phylogenetic analyses were performed to determine the microbial changes between the two conditions. Results indicated the predominant bacterial populations present in the rumen were comprised of the phyla Firmicutes and Bacteroidetes, irrespective of presence/absence of TNT in the diet. Significant differences (P < 0.001) were found between the community structure of the bacteria under TNT (-) and TNT (+) diets. Examination of the TNT (+) diet showed an increase in the clones belonging to family Ruminococcaceae, which have previously been shown to degrade TNT in pure culture experiments.

Acute aquatic toxicity of western juniper (Juniperus occidentalis) foliage and Port Orford cedar (Chamaecyparis lawsoniana) heartwood oils.

Recently, interest has developed for using essential oils from Western juniper (Juniperus occidentalis) foliage and Port Orford cedar (Chamaecyparis lawsoniana) heartwood in commercial products such as pest repellents and cosmetics. In order to gauge the relative toxicological risk that these oils pose to freshwater and marine organisms, the acute aquatic toxicity of these oils was evaluated using OPPTS guidelines to the cladoceran Daphnia magna, the rainbow trout Oncorhynchus mykiss and the green alga Selenastrum capricornutum. For western juniper foliage oil, no toxicity was exhibited toward D. magna or O. mykiss, even at 5.0 mg/L (the highest concentration tested and limit of solubility). For toxicity to S. capricornutum using algal cell density, the 72 and 96 h EC50 value was 1.7 mg/L and the no observable effect concentration (NOEC) was 0.63 mg/L. For Port Orford cedar heartwood oil, no toxicity was exhibited toward O. mykiss or S. capricornutum, even at 5.0 mg/L (the highest concentration tested and limit of solubility). The 48-h D. magna EC50 value was 1.9 mg/L; the NOEC values for algal cell density were 1.25 mg/L (72 h) and 0.63 mg/L (96 h). In summary, this study shows that western juniper foliage and Port Orford cedar heartwood oils demonstrate little to no risk to aquatic organisms.

Microbiomic comparison of the intestine of the earthworm Eisenia fetida fed ergovaline.

Tall fescue toxicosis and ergot alkaloids cost U.S. livestock producers approximately one billion dollars in annual livestock production loss annually. Ergovaline (EV) is the tall fescue alkaloid primarily responsible for clinical disease in livestock. Since native ruminal microorganisms have not been attributed to the detoxification of EV, finding detoxifying microbes from other environments is desirable. One possible source for potential microorganisms that can degrade EV is the anaerobic gut of the earthworm, Eisenia fetida. This study describes a comparative microbial analysis of earthworm digestive tracts receiving 10,000 ppb EV (E+ treatment) when compared with a control treatment with no detectable amounts of EV (E- treatment). An HPLC assay determined a 25% loss of EV from the E+ treatment was microbial in nature. A community microbiomic approach of constructing 16S-rRNA gene clone libraries was used to compare the microbes affected by the two treatments. RDPII tools such as Classifier and Libcompare were used in the analysis of 16S sequences. DOTUR analysis was used to examine the richness and diversity of the two microbial populations in these experiments. The results indicate there are few significant differences in the microbial community structure between the two microbiomes.

Molecular characterization of sheep ruminal enrichments that detoxify pyrrolizidine alkaloids by denaturing gradient gel electrophoresis and cloning.

An enrichment of strictly anaerobic bacteria from ovine rumen fluid, which has previously been named L4M2, is known to detoxify animal hepatotoxins from the pyrrolizidine alkaloid family. These toxins are present in the tansy ragwort plant (Senecio jacobaea). These plants have been described in livestock animals' range forages in regions of the world such as the Northwest United States and South Africa. The bacterial enrichment was characterized by molecular cloning techniques and by the molecular fingerprinting technique of denaturing gradient gel electrophoresis (DGGE). Phylogenetic analysis of the enrichment revealed that the consortium is composed of no more than five putative bacterial species which associated to the Anaerovibrio, Desulfovibrio, Megasphaera, Prevotella, and Synergistes generas. These are all known to exist in the upper gastrointestinal tract of ruminant animals. This work improved upon previous attempts to characterize the consortium by obtaining nearly full-length ribosomal 16S rDNA sequences through cloning. The DGGE results were directly compared to the cloning data by polymerase chain reaction (PCR) amplifying eight phylogenetically representative clones and analyzing them by DGGE. Direct DGGE analysis of the enrichment displayed greater 16S diversity than the clone library used in this study, suggesting that at least one of the organisms present in the enrichment comprises less than 1% of the total cell population. These data will be used to further refine the enrichment in hopes of future use as a probiotic, which could be administered to animals challenged by the presence of tansy ragwort in their forage.

Assessment of serum concentrations and sedative effects of fentanyl after transdermal administration at three dosages in healthy llamas.

OBJECTIVE: To determine the serum concentrations and sedative effects of fentanyl after transdermal administration at 3 dosages in llamas. ANIMALS: 9 healthy adult female llamas (mean age, 8 +/- 3 years; mean weight, 150 +/- 18 kg). PROCEDURE: Llamas were allocated to 1 of 3 groups (3 llamas/group). Fentanyl patches (each providing transdermal delivery of 75 microg of fentanyl/h) were placed on shaved areas of the antebrachium of all llamas. In group 1, llamas were treated with 1 patch (anticipated fentanyl dosage, 75 microg/h). In group 2, llamas were treated with 2 patches (anticipated fentanyl dosage, 150 microg/h). In group 3, llamas were treated with 4 patches (anticipated fentanyl dosage, 300 microg/h). For each llama, the degree of sedation was assessed by use of a subjective scoring system and a blood sample was collected for determination of serum fentanyl concentration at 12, 24, 36, 48, 60, and 72 hours after patch placement. RESULTS: Following the placement of 4 patches, mean +/- SD serum fentanyl concentration in group 3 llamas reached 0.3 +/- 0.08 ng/mL within 12 hours. This concentration was sustained for 72 hours. In group 2, application of 2 patches provided inconsistent results; in group 1, application of 1 patch rarely provided measurable serum fentanyl concentrations. No llamas became sedated at any time. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that application of four 75 microg/h fentanyl patches provides consistent, sustained serum fentanyl concentrations without sedation in llamas. However, the serum concentration of fentanyl that provides analgesia in llamas is not known.

Comparison of hepatic in vitro metabolism of the pyrrolizidine alkaloid senecionine in sheep and cattle.

OBJECTIVE: To compare hepatic metabolism of pyrrolizidine alkaloids (PAs) between sheep and cattle and elucidate the protective mechanism of sheep. SAMPLE POPULATION: Liver microsomes and cytosol from 8 sheep and 8 cattle. PROCEDURE: The PA senecionine, senecionine N-oxide (nontoxic metabolite) and 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP; toxic metabolite) were measured in microsomal incubations. The kcat (turnover number) was determined for DHP and N-oxide formation. Chemical and immunochemical inhibitors were used to assess the role of cytochrome P450s, flavin-containing monooxygenases (FMOs), and carboxylesterases in senecionine metabolism. The CYP3A, CYP2B, and FMO concentrations and activities were determined, in addition to the role of glutathione (GSH) in senecionine metabolism. RESULTS: DHP concentration did not differ between species. Sheep formed more N-oxide, had higher N-oxide kcat, and metabolized senecionine faster than cattle. The P450 concentrations and isoforms had a large influence on DHP formation, whereas FMOs had a large influence on N-oxide formation. In cattle, CYP3A played a larger role in DHP formation than in sheep. FMO activity was greater in sheep than in cattle. Addition of GSH to in vitro microsomal incubations decreased DHP formation; addition of cytosol decreased N-oxide formation. CONCLUSIONS AND CLINICAL RELEVANCE: Hepatic metabolism differences alone do not account for the variation in susceptibility seen between these species. Rather, increased ruminal metabolism in sheep appears to be an important protective mechanism, with hepatic enzymes providing a secondary means to degrade any PAs that are absorbed from the rumen.

Toxicity studies on western juniper oil (Juniperus occidentalis) and Port-Orford-cedar oil (Chamaecyparis lawsoniana) extracts utilizing local lymph node and acute dermal irritation assays.

The essential oil extracts of western juniper oil (Juniperus occidentalis) and Port-Orford-cedar oil (Chamaecyparis lawsoniana) were evaluated for possible dermal toxic effects on mice and rabbits. Mice were tested for their response to both extracts utilizing a local lymph node assay. Western juniper oil extract at 0.5% and 5% concentrations did not show a stimulation index (SI) greater than normal (3.0); however, a 50% concentration did show a positive response at 3.3. Port-Orford-cedar oil extract did not show a positive response at concentrations of 0.5%, 5% or 50%. An acute dermal irritation study using rabbits had a primary irritation index (PII) of 3.3 with 100% Port-Orford-cedar oil extract. This was reduced to a PII of 0.625 when diluted 1:1 with olive oil. Undiluted western juniper oil extract had a PII score of 2.7. While a 5.0% solution had a PII score of 0.3, a 0.5% solution of western juniper oil was a non-irritant. It would appear that animals bedded on wood shavings have contact with essential oils at concentrations far less than the 2% maximum by weight obtained by steam distillation extraction. These concentrations did not elicit a hypersensitivity response.

Metabolism of pyrrolizidine alkaloids by Peptostreptococcus heliotrinreducens and a mixed culture derived from ovine ruminal fluid.

A mixed culture of ovine ruminal microbes metabolizes the macrocyclic pyrrolizidine alkaloids present in the plant Senecio jacobaea, including jacobine and seneciphylline. Previous attempts to identify metabolites of these alkaloids have not been successful. The objective of this study was to compare the metabolism of pyrrolizidine alkaloids by a mixed culture of ovine ruminal microbes to the metabolism of pyrrolizidine alkaloids by the known organism Peptostreptococcus heliotrinreducens. P. heliotrinreducens metabolizes the pyrrolizidine alkaloids heliotrine and lasiocarpine to 7alpha-hydroxy-1-methylene-8alpha-pyrrolizidine and 7alpha-angelyl-1-methylene-8alpha-pyrrolizidine, respectively. This organism does not metabolize the pyrrolizidine alkaloids jacobine or seneciphylline. A mixed culture of ovine ruminal microbes also metabolized heliotrine and lasiocarpine to identical methylene compounds. This mixed culture also metabolized jacobine and seneciphylline, with the production of very low levels of the corresponding 1-methylene compounds. Samples were analyzed by TLC and GC/MS.