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Epoxyjanthitrems I-IV ( 1 - 4 ) and epoxyjanthitriol ( 5 ) were isolated from seed of perennial ryegrass ( Lolium perenne ) infected with the endophytic fungus Epichloë festucae var. lolii. Although structures for epoxyjanthitrems I-IV have previously been proposed in the literature, this is the first report of a full structural elucidation yielding NMR (Nuclear magnetic resonance) assignments for all five epoxyjanthitrem compounds, and additionally, it is the first isolation of epoxyjanthitriol ( 5 ). Epoxyjanthitrem I induced tremors in mice and gave a dose dependent reduction in weight gain and feeding for porina ( Wiseana cervinata ), a common pasture pest in New Zealand. These data suggest that epoxyjanthitrems are involved in the observed effects of the AR37 endophyte on livestock and insect pests.

Aims: To compare ryegrass pastures infected with endophytes producing diverse alkaloids for their ability to cause ryegrass staggers in grazing lambs; to compare respiration rates and rectal temperatures of these lambs after exposure to heat stress, and to compare liveweight gains during the study period., Methods: Ryegrass pastures of cultivar Trojan infected with NEA endophytes, branded NEA2 (T-NEA2), endophyte-free Trojan (T-NIL), Samson infected with standard endophyte (S-STD), Samson infected with AR37 endophyte (S-AR37) and endophyte-free Samson (S-NIL), were grazed by lambs (n=30 per cultivar) for up to 48 days in February and March of 2012 and 2013. Pasture samples were analysed for alkaloid concentrations and lambs were scored for ryegrass staggers at intervals during the study period. Liveweight was recorded at the start (Day 0) and end of the study, and rectal temperatures and respiratory rates were measured in lambs exposed to heat stress on Days 23 and 26, in 2012 and 2013, respectively., Results: Concentrations of alkaloids were lower in 2012 than 2013, associated with warmer and drier conditions in 2013, and the prevalence of ryegrass staggers was low in 2012. In 2013, concentrations of ergovaline were similar in T-NEA2 and S-STD, but concentrations of lolitrem B were lower in T-NEA2 than S-STD. S-AR37 produced epoxy-janthitrems but no lolitrem B or ergovaline. In 2013, by Day 20, 9/30 (30%) sheep grazing S-STD had severe staggers (score ≥4), and by Day 47 all sheep had been removed from this cultivar due to severe staggers. By Day 47, 18/30 (60%), 4/30 (13%) and 0/30 (0%) sheep grazing S-AR37, T-NEA2 and T-NIL pastures, respectively, had severe staggers. There were no differences in mean daily weight gain of lambs between cultivars in either year. In both years, mean rectal temperature and respiration rate following exposure to heat stress were highest in sheep grazing S-STD and T-NEA2, and lowest in sheep grazing T-NIL., Conclusions: In lambs grazing different ryegrass pastures infected with endophytes, ryegrass staggers was most severe on S-STD, less severe on S-AR37 and least on T-NEA2. When under heat stress, lambs grazing ergovaline-producing S-STD and T-NEA2 pastures had increased respiration rates and rectal temperatures compared with lambs grazing T-NIL., Clinical Relevance: If ambient temperatures are suitable, NEA2-branded endophytes have the potential to express concentrations of ergovaline sufficient to induce heat stress in grazing sheep.

Aim: To determine the concentrations of fungal endophyte toxins in the milk of cows fed perennial ryegrass containing wild-type or AR37 endophyte., Methods: Groups of 10 multiparous Holstein-Friesian cows were fed wild-type (containing lolitrem B) or AR37 (containing epoxy-janthitrems) endophyte-infected perennial ryegrass (Lolium perenneL.). Animals were kept indoors and fed for 12 days. Over this period, animals were regularly assessed for ryegrass staggers and herbage intake measured. At the conclusion of the 12-day indoor-feeding period, cows were grazed on AR1 (toxin-free) pastures for a further 8 days. Daily individual milk samples and milk yields were collected over the complete 20-day period. Milk samples were analysed for endophyte toxins using HPLC methods developed during this study. Daily herbage samples were also taken and concentrations of endophyte toxins measured., Results: Methods were successfully developed for the analysis of lolitrem B and epoxy-janthitrems in milk which allowed the concentrations of these compounds in milk to be compared with the concentrations in feed consumed by the animals. Both toxin types could be detected in milk after only 1 day of exposure to respective treatment pastures. The maximum concentration of endophyte toxins in milk was 5 ng/mL lolitrem B and 109 ng/mL epoxy-janthitrems from cows fed wild-type and AR37 endophyte-infected ryegrass pastures, respectively. Concentrations of epoxy-janthitrems present in herbage were much higher than for lolitrem B (Day 1-12 average of 14.6 and 1.8 ppm, respectively). Despite the high concentrations of epoxy-janthitrems consumed by cows fed AR37 endophyte-infected pastures no signs of ryegrass staggers were observed over the experimental period, whereas those cows fed wild-type endophyte-infected pastures all showed signs of ryegrass staggers. This is consistent with the view that epoxy-janthitrems are low potency tremorgens. At the conclusion of the toxin feeding period, endophyte toxin concentrations in milk quickly dropped to almost zero after 8 days. A comparison of the quantities of lolitrem B and epoxy-janthitrems consumed by each cow with the quantities secreted in milk showed that only very low proportions of the total amount ingested are secreted in milk (0.23% lolitrem B and 0.49% epoxy-janthitrems)., Conclusion: Lolitrem B and epoxy-janthitrems can be detected in the milk of cows consuming wild-type and AR37 endophyte-infected perennial ryegrass, respectively. Concentrations detected were low and changed quickly in association with the amounts being consumed by the cows. Available evidence gives no indication that these compounds may pose a threat to human health.

Aim: To monitor changes in concentrations of lolitrem B and epoxy-janthitrems in the fat of sheep grazing perennial ryegrass infected with wild-type- and AR37-endophyte, respectively, during the time of year when ryegrass staggers would be expected to be observed., Methods: Ten 5-month-old lambs with no previous exposure to endophytes were grazed on either wild-type (containing lolitrem B, n = 5) or AR37 (containing epoxy-janthitrems, n = 5) endophyte-infected perennial ryegrass (Lolium perenne L.) pastures between October 2008 and June 2009. Animals were regularly assessed for ryegrass staggers using the Keogh scale (0 = no signs, 5 = severe tremors). When a score of > 3.5 was observed animals were removed from the treatment pastures for 1 month. Fat biopsy samples were taken from each animal at approximately monthly intervals and analysed for endophyte metabolites using high performance liquid chromatography (HPLC) methods developed during this study. Regular herbage samples were also taken and concentrations of endophyte metabolites measured., Results: Efficient and reproducible methods to analyse both lolitrem B and epoxy-janthitrems in fat were developed. Concentrations of lolitrem B and epoxy-janthitrems in herbage and in sheep fat increased from late November to peak in mid-February. Ryegrass staggers was observed in both groups of sheep at this time. Following 1 month of grazing non-infected pasture mean concentrations in fat of lolitrem B decreased by 43% from 61.8 to 35.3 ppb, and of epoxy-janthitrems by 38% from 1032.0 to 639.5 ppb. Maximum concentrations in herbage of epoxy-janthitrems (35.7 ppm) were higher than of lolitrem B (3.4 ppm), but signs of staggers were less severe in sheep grazing pasture containing the former compared with the latter (median Keogh scores in late February were 2 and 3, respectively), consistent with epoxy-janthitrems being low potency toxins., Conclusion: This study demonstrated that concentrations of epoxy-janthitrems and lolitrem B in sheep fat increased quickly during the initial phase of the study when concentrations in pasture increased, and decreased when animals were removed from pastures containing these compounds. These data will be used in the risk assessment of the endophyte metabolites.

AIM: To investigate a possible interaction between lolitrem B and ergovaline by comparing the incidence and severity of ryegrass staggers in sheep grazing ryegrass (Lolium perenne) containing lolitrem B or ryegrass containing both lolitrem B and ergovaline. METHODS: Ninety lambs, aged approximately 6 months, were grazed on plots of perennial ryegrass infected with either AR98 endophyte (containing lolitrem B), standard endophyte (containing lolitrem B and ergovaline) or no endophyte, for up to 42 days from 2 February 2010. Ten lambs were grazed on three replicate plots per cultivar. Herbage samples were collected for alkaloid analysis and lambs were scored for ryegrass staggers (scores from 0–5) weekly during the study. Any animal which was scored ≥4 was removed from the study. RESULTS: Concentrations of lolitrem B did not differ between AR98 and standard endophyte-infected pastures during the study period (p=0.26), and ergovaline was present only in standard endophyte pastures. Ryegrass staggers was observed in sheep grazing both the AR98 and standard endophyte plots, with median scores increasing in the third week of the study. Prior to the end of the 42-day grazing period, 22 and 17 animals were removed from the standard endophyte and AR98 plots, respectively, because their staggers scores were ≥4. The cumulative probability of lambs having scores ≥4 did not differ between animals grazing the two pasture types (p=0.41). CONCLUSIONS AND CLINICAL RELEVANCE: There was no evidence for ergovaline increasing the severity of ryegrass staggers induced by lolitrem B. In situations where the severity of ryegrass staggers appears to be greater than that predicted on the basis of concentrations of lolitrem B, the presence of other tremorgenic alkaloids should be investigated. [ABSTRACT FROM PUBLISHER]