Ergovaline does not alter the severity of ryegrass staggers induced by lolitrem B.

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.

Pathological changes seen in horses in New Zealand grazing Mediterranean tall fescue (Lolium arundinaceum) infected with selected endophytes (Epichloë coenophiala) causing equine fescue oedema.

AIM: To investigate whether Mediterranean tall fescue (Lolium arundinaceum (Schreb.) Darbysh. (syn Festuca arundinacea)) infected with selected fungal endophytes (Epichloë coenophiala (formerly Neotyphodium coenophialum)) caused equine fescue oedema when grown in New Zealand, and to examine the pathological changes associated with this intoxication. METHODS: Horses were grazed on Mediterranean tall fescue that was infected with the endophytes AR542 (n=2), or AR584 (n=3), or Mediterranean tall fescue that was endophyte-free (n=2). Blood samples were taken up to 7 days after the start of feeding to detect changes in concentrations of total protein in serum and packed cell volume. Any horse showing clinical evidence of disease was subject to euthanasia and necropsy. RESULTS: Within 6 days, both horses grazing fescue infected with AR542 became depressed and lethargic. One horse grazing fescue infected with endophyte AR584 became depressed within a 5-day feeding period while another horse in this group died shortly after being removed from the AR584 pasture. The third horse in this group did not develop clinical signs within the 5-day feeding period. However, haemoconcentration and hypoproteinaemia was detected in all horses grazing Mediterranean tall fescue that was infected by AR542 or AR584 endophyte. No abnormalities were observed in horses grazing fescue that was endophyte-free. Necropsy examination was performed on two horses grazing fescue infected with AR542 and one horse grazing fescue infected with AR584. All three horses had marked oedema of the gastrointestinal tract. Histologically, the oedema was accompanied by large numbers of eosinophils, but no necrosis. CONCLUSIONS: Horses grazing Mediterranean tall fescue that was infected by AR542 or AR584 developed hypoproteinaemia and haemoconcentration, most likely due to leakage of plasma proteins into the gastrointestinal tract. This suggests that these selected endophytes produce a compound that is toxic to horses, although the toxic principle is currently unknown. CLINICAL RELEVANCE: Mediterranean tall fescue that is infected by AR542 or AR584 should not be fed to horses in New Zealand. This intoxication should be considered in horses in which a rapid onset of hypoproteinaemia and haemoconcentration is detected. This intoxication should also be considered if marked gastrointestinal oedema is observed.

Further investigation of equine fescue oedema induced by Mediterranean tall fescue (Lolium arundinaceum) infected with selected fungal endophytes (Epichloë coenophiala).

AIMS To determine if equine fescue oedema (EFO) induced by grazing Mediterranean-type tall fescue (Lolium arundinaceum) infected with selected endophytes (Epichloë coenophiala) could be prevented by treatment with the corticosteroid, methylprednisolone, and anti-histamine, cetirizine, and to determine concentrations of lolines, specifically N-acetyl norloline (NANL), in grasses grazed by horses that did and did not develop EFO. METHODS Four horses were grazed on AR542-infected Mediterranean tall fescue pasture (from Day 0) for 7 days prior to being subjected to euthanasia. Two of these horses were treated with 250 mg methylprednisolone and 300 mg cetirizine hydrochloride every 12 hours orally from Days 0-7. Two more horses grazed meadow fescue (Festuca pratensis) infected with the naturally-occurring, common endophyte (Epichloë uncinata) for 21 days before euthanasia. All horses were observed closely for signs of EFO, and blood samples were taken daily for measurement of concentrations of total protein (TP) in serum. Following euthanasia post-mortem examinations were conducted on all horses. Pasture samples of meadow fescue and Mediterranean tall fescue from the current study, and endophyte-infected Mediterranean tall fescue from a previous study that were associated with EFO, were analysed for concentrations of lolines using gas chromatography. RESULTS By Day 7, the treated and untreated horses grazing AR542-infected Mediterranean tall fescue all developed signs of EFO, and concentrations of TP in serum of all horses were <45 g/L. No signs of EFO were observed in horses grazing meadow fescue and concentrations of TP remained above 60 g/L. Necropsy showed marked oedema and eosinophilic inflammation in the intestines of all horses grazing Mediterranean tall fescue. In the sample of meadow fescue, concentrations of total lolines and N-acetyl norloline (NANL) were 2,402 and 543 mg/kg, respectively. In the three samples of Mediterranean tall fescue associated with EFO, concentrations of total lolines were 308, 629 and 679 mg/kg, and concentrations of NANL were 308, 614 and 305 mg/kg. CONCLUSIONS AND CLINICAL RELEVANCE In horses grazing Mediterranean tall fescue infected with the AR542 endophyte, treatment with methylprednisolone and cetirizine did not prevent development of EFO. Results of pasture analysis indicated that lolines or NANL are unlikely to be the causative agent of this disease.

The occurrence of ryegrass staggers and heat stress in sheep grazing ryegrass-endophyte associations with diverse alkaloid profiles.

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.

The evaluation of endophyte toxin residues in sheep fat.

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.

Clinical expression of lolitrem B (perennial ryegrass) intoxication in horses.

REASONS FOR PERFORMING STUDY: Perennial ryegrass staggers is purported to be a common neurological mycotoxicosis of horses but the case description lacks detail and evidence. OBJECTIVE: To describe the clinical syndrome of lolitrem B intoxication in horses, limiting tests to those that are applicable to clinical practice, and to assess the potential value of enzyme-linked immunosorbent assay (ELISA) tests for lolitrem B in horse body fluids. METHODS: Seven horses in 2 separate groups were fed perennial ryegrass seed and hay containing 2 ppm lolitrem B. Paired data were collected prior to and after 2 weeks exposure to lolitrem B, including video-documented neurological examination and clinical examination. RESULTS: All horses developed a variable degree of tremor and ataxia when exposed to lolitrem B. Tremor depended on the level of activity and included a subtle, rapid tremor of the eyeball. Ataxia was exaggerated by blindfolding and primarily involved a truncal sway and irregular, but predictable, limb placements. No change was detected in urine lolitrem B levels and, although plasma lolitrem B increased during the treatment period, levels did not correlate with the severity of clinical signs displayed. Limb swelling, heel lesions and serous nasal discharge were also observed in horses most severely intoxicated. CONCLUSIONS: The clinical effects of lolitrem B intoxication in horses primarily involve action-related tremors and symmetrical vestibular ataxia. Ergovaline may have caused the limb swelling, heel lesions and serous nasal discharge. Plasma ELISA for lolitrem B may be of diagnostic use in the future. POTENTIAL RELEVANCE: This study provides a clearer appreciation of the clinical signs and variability of perennial ryegrass intoxication in horses.

Excretion of loline alkaloids in urine and faeces of sheep dosed with meadow fescue (Festuca pratensis) seed containing high concentrations of loline alkaloids.

AIM: To determine the effect of oral dosing of sheep with loline alkaloids on their excretion in urine and faeces, and to monitor for any toxic effects. METHODS: In Experiment 1, six 9-month-old ewe lambs were given a single oral dose of loline alkaloids (52 mg/kg bodyweight (BW); acute exposure) as a suspension of ground meadow fescue (Festuca pratensis) seed in water. In Experiment 2, on six consecutive days, six ewe lambs were given three doses of loline (68 mg/kg BW/day; chronic exposure). Blood was collected at variable intervals up to 72 h in Experiment 1, and up to 8 days in Experiment 2, for haematology and measurement of alkaline phosphatase, aspartate aminotransaminase, creatine kinase and γ-glutamyl transferase in plasma. Urine and faecal samples were collected at similar times for measurement of creatinine in urine and loline alkaloid analysis. A post mortem with histopathology was carried out on two animals at the end of each experiment. RESULTS: The loline alkaloids, N-acetyl norloline, N-formyl loline, N-acetyl loline, N-methyl loline and loline base were detected in urine within 15 minutes after the single dosing. N-formyl loline and loline base were the predominant metabolites in urine in both experiments. The total quantity of lolines excreted in both urine and faeces was 10% and 4% of the amount dosed in Experiments 1 and 2, respectively. In both experiments, the clinical chemistry parameters in blood and urine were within normal ranges. Post-mortem and histopathological examination did not show any abnormalities. CONCLUSIONS: This is the first report of loline alkaloid profiles in both urine and faeces of sheep. The appearance of loline alkaloids and the loline base in urine within 15 minutes suggests rapid uptake, metabolism and excretion. Loline alkaloids were non-toxic to sheep at the concentrations they are exposed to under New Zealand grazing conditions. The low recovery of loline alkaloids in urine and faeces in the absence of toxicity signs suggests lolines are extensively metabolised; probably to forms other than N-formyl loline, N-methyl loline, N-acetyl loline, N-acetyl norloline, and loline base in the digestive tract of sheep prior to absorption, and/or in the liver or other tissues following absorption.

Haemodynamics of lambs grazing perennial ryegrass (Lolium perenne L.) either infected with AR6 novel, wild-type endophyte, or endophyte-free.

AIM: To compare vasoconstriction of the auricular artery and characteristics of blood flow in the carotid arteries between lambs grazing perennial ryegrass that was either infected with the AR6 novel endophyte (AR6), wild-type endophyte or endophyte-free. METHODS: Sixteen Coopworth lambs, mean 23.7 (SD 1.8) kg, were randomly assigned to graze three, 0.10-ha pastures of perennial ryegrass (cultivar Extreme) located in Lincoln, New Zealand, that were infected with either the AR6 novel endophyte (n=5) or wild-type endophyte (n=6) or were endophyte-free (n=5), for 18 days until 16 March 2009. Lambs on AR6 pasture were then switched to endophyte-free pasture, and those on endophyte-free were switched to AR6 pasture, for 18 days. Lambs continued grazing the wild-type ryegrass during both phases of the study. Colour Doppler ultrasonography was used to monitor cross-sectional area of lumina in the auricular and carotid arteries as measurements of vasoconstriction, and to measure pulsatility indices, heart rate, systolic and diastolic velocities, and mean velocity in the carotid artery. Urine was sampled once during each phase, to measure the concentration of urinary alkaloids. A heat challenge (32°C) was imposed on the last day of the experiment, to determine treatment effects on rectal temperature and respiration rate. RESULTS: Vasoconstriction was detected in the auricular arteries of lambs grazing AR6 and wild-type pastures. Areas of lumina declined linearly over 9 days in lambs that were switched from endophyte-free to AR6 pastures (p<0.05), and areas increased linearly in lambs that were switched from AR6 to endophyte-free pasture (p<0.001). Resistance to blood flow in the carotid arteries decreased linearly in the 9 days after lambs were switched from AR6 to endophyte-free pasture(p<0.05), and tended to increase after lambs on endophyte-free pasture were switched to AR6 (p<0.10). The concentrations of urinary alkaloids decreased after switching lambs from AR6 to endophyte-free pasture, and increased after switching from endophyte-free to AR6 pasture (p<0.05). The concentrations of urinary alkaloids of lambs on wild-type pasture were similar between the conditioning and experimental phases. There were no treatment effects on rectal temperature and respiration rate during the heat challenge. CONCLUSIONS: These results indicated that management approaches are needed to overcome potential vulnerabilities to heat stress for sheep grazing AR6 perennial ryegrass. Furthermore, following grazing such pasture, lambs will need to graze endophyte-free ryegrass for >18 days, to completely clear ergot alkaloids from their vasculature, assuming that complete clearance can be achieved.

Modifying diets to satisfy nutritional requirements using linear programming.

A computational method for constructing individually acceptable diets by modifying a chosen diet to meet nutritional requirements is described. The effects on food quantities of imposing different nutrient requirements on a sample diet are demonstrated and techniques which can ensure the acceptability to the individual of the modified diet are described. The starting point in the calculation is the person's current dietary intake. This is modified using linear programming methods which make the smallest changes to the food quantities to meet specific targets. Sequential modification can be used to identify changes that are acceptable to the individual. The computer program has been developed in collaboration with practising dietitians and is in use in some leading UK hospitals.

Prediction of a glucose appearance function from foods using deconvolution.

The glycaemic response of an insulin-treated diabetic patient goes through many transitory phases, leading to a steady state glycaemic profile following a change in either insulin regimen or diet. Most models attempting to model the glucose and insulin relationship try to model the effect of oral or injected glucose rather than that from the digestion of food. However, it is clear that a better understanding of the glycaemic response would arise from consideration of intestinal absorption from the gut. It is assumed that this type of absorption can be modelled by a so-called glucose appearance function (systemic appearance of glucose via glucose absorption from the gut) predicting the glucose load from the food. Much research has been carried out in the areas of hepatic balance, insulin absorption and insulin independent/dependent utilization. However, little is known about intestinal absorption patterns or their corresponding glucose appearance profiles. The strategy under investigation herein is to use deconvolution or backward engineering. By starting with specific results i.e. blood glucose and insulin therapy, it is possible to work backwards to predict the glucose forcing functions responsible for the outcome. Assuming compartmental consistency, this will allow a clearer insight into the true gut absorption process. If successful, the same strategy can be applied to more recent glucose and insulin models to further our understanding of the food to blood glucose problem. This paper investigates the Lehmann-Deutsch modified model of glucose and insulin interaction, created from the model proposed by Berger-Rodbard. The model attempts to simulate the steady state glycaemic and plasma insulin responses, independent of the initial values from which the simulation is started. Glucose enters the model via both intestinal absorption and hepatic glucose production. We considered a 70 kg male insulin-dependent diabetic patient with corresponding hepatic and insulin sensitivity parameters of 0.6 and 0.3 respectively. Net hepatic glucose balance was modelled piecewise by linear and symmetric functions. A first-order Euler method with step size of 15 minutes was employed. For the simulation, only Actrapid and NPH injections were considered. The injection of insulin and the glucose flux to the gut were started simultaneously to avoid any delay associated with gastric emptying. The systemic appearance of glucose was compared from two view points, not only to assess the strategic principle, but also to assess the suitability of the modifications made by Lehmann and Deutsch. The first is a forward prediction using the compartmental structure. This analysis involves the rate of gastric emptying without time delay. The second is a backward prediction from experimentally observed blood glucose profiles. Investigations involved porridge, white rice and banana containing the same carbohydrate content (25 g). Results obtained from the first analysis were dependent on the rate of gastric emptying, especially its ascending and descending branches. Results from the second analysis were dependent on the dose and type of insulin administered. Both predicted profiles showed consistency with physiological reasoning, although it became apparent that such solutions could be unstable. Furthermore, both types of prediction were similar in structure and appearance, especially in simulations for porridge and banana. This emphasized the consistency and suitability of both analyses when investigating the compartmental accuracy and limitations within a model. The new strategic approach was deemed a success within the model, and the modifications made by Lehmann and Deutsch appropriate. We suggest that a gastric emptying curve with a possible gastric delay is the way forward in regulating the appearance of glucose via gut absorption. The Lehmann-Deutsch gastric curve is described by either a trapezoidal or triangular function dependent on the carbohydrate cont