Lolitrem B residue in fat tissues of cattle consuming endophyte-infected perennial ryegrass straw.

Lolitrems are neurotoxins found in endophyte-infected perennial ryegrass. Lolitrems, primarily lolitrem B, are the causative agents of ryegrass staggers in livestock. To guarantee the safety of meat produced from cattle consuming endophyte-infected perennial ryegrass, lolitrem B concentrations in tissues of Japanese Black cattle were determined using high-performance liquid chromatography. Lolitrem B was not detected in muscle, liver, kidney, or cerebrum of a Japanese Black cow with signs of ryegrass staggers. In contrast, perirenal fat contained 210 ppb lolitrem B. Three cows that received half as much perennial ryegrass straw as the cow with ryegrass staggers showed no clinical signs of ryegrass staggers. However, low concentrations of lolitrem B (less than 150 ppb) were detected in their fat tissue. These observations indicate that human exposure to the neurotoxic effect of lolitrem B through beef is unlikely. The amount of lolitrem B consumed by cattle can be estimated by the determination of lolitrem B in fat tissue.     

Correlation of ergovaline and lolitrem B levels in endophyte-infected perennial ryegrass (Lolium perenne).

The varieties of perennial ryegrass (Lolium perenne) infected with the endophytic fungus Neotiphodium lolii contain several classes of toxic alkaloids, including ergopeptide alkaloids and lolitrem alkaloids. Lolitrem B, a potent tremorgen, is generally considered to be the predominant alkaloid in endophyte-infected perennial ryegrass. Ergovaline, a vasoconstrictor normally associated with endophyte-infected tall fescue (Festuca arudinacea), is also present in endophyte infected perennial ryegrass. Clinical signs of animals ingesting endophyte-infected perennial ryegrass are consistent with the presence of lolitrem B. However, clinical signs normally associated with ergovaline poisoning are not usually observed in animals ingesting endophyte-infected perennial ryegrass. A survey was conducted to quantitate both lolitrem B and ergovaline in 459 perennial ryegrass straw samples received at the Oregon State University College of Veterinary Medicine. Samples were analyzed for each alkaloid using separate high-performance liquid chromatography analyses. A strong positive correlation between the 2 alkaloids (r2 = 0.7335) was observed, especially in the samples containing <3,000 ppb (ng/g) lolitrem B. The threshold levels above which clinical signs typically occur are 2,000 ppb lolitrem B and 300-400 ppb ergovaline. All of the samples analyzed contained <425 ppb ergovaline.     

Evaluation of perennial ryegrass straw as a forage source for ruminants

Two experiments were conducted to evaluate perennial ryegrass straw as a forage source for ruminants. Experiment 1 evaluated digestion and physiological variables in steers offered perennial ryegrass straw containing increasing levels of ergot alkaloid, lolitrem B. Sixteen ruminally cannulated Angus x Hereford steers (231+/-2 kg BW) were blocked by weight and assigned randomly to one of four treatments. Steers were provided perennial ryegrass straw at 120% of the previous 5-d average intake. Before straw feeding, soybean meal was provided (0.1% BW; CP basis) to meet the estimated requirement for degradable intake protein. Low (L) and high (H) lolitrem B straws (<100 and 1,550 ppb, respectively; DM basis) were used to formulate treatment diets: 100% L; 67% L:33% H; 33% L:67% H; 100% H (DM basis). Intake and digestibility of DM and OM, and ruminal pH, total VFA, and NH3-N were not affected by increasing lolitrem B concentration. Ruminal indigestible ADF (IADF) fill increased linearly (P = 0.01) and IADF passage rate decreased linearly (P = 0.04) as lolitrem B increased. Experiment 2 evaluated performance and production by 72 Angus x Hereford cows (539+/-5 kg BW) consuming perennial ryegrass straw containing increasing lolitrem B during the last third of gestation. Cows were blocked by body condition score and randomly assigned to one of three treatments. Cows were provided perennial ryegrass straw ad libitum and supplemented with soybean meal (0.1% BW; CP basis) to meet the estimated requirement for degradable intake protein. Mixtures of a L and H lolitrem B straw (467 and 2,017 ppb, respectively) were used to formulate treatment diets: 100% L, 50% L:50% H, 100% H (DM basis). Thirteen of 24 cows on the 100% H treatment exhibited signs of ryegrass staggers and were removed from the study; nevertheless, lolitrem B concentration did not influence pre- or postcalving weight or body condition score change. These data suggest that feeding perennial ryegrass straw containing up to 1,550 ppb lolitrem B (DM basis) did not adversely affect nutrient digestion or physiological response variables in steers. However, providing straw with a lolitrem B concentration of approximately 2,000 ppb (DM basis) resulted in 54% of cows exhibiting signs of ryegrass staggers. These data suggest that blending straws with a high (>2,000 ppb) and low (<500 ppb) concentration of lolitrem B can be a successful management practice.     

The evaluation of endophyte toxin residues in sheep fat

Abstract

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.     

The effect of glyphosate, paraquat and paclobutrazol on lolitrem B levels in endophyte-infected perennial ryegrass

Two herbicides (glyphosate and paraquat) and a plant growth regulator (paclobutrazol) were applied to endophyteinfected (Acremonium lolii) perennial ryegrass swards. Subsamples of these swards were then chemically analysed at intervals up to 28 days later for lolitrem B, the compound responsible for perennial ryegrass staggers in domestic livestock. Glyphosate and paclobutrazol had no effect on lolitrem B concentrations. Paraquat applications decreased lolitrem B concentrations in the herbage. Because none of the chemicals tested increased the concentration of lolitrem B in the herbage, they are unlikely to be directly implicated in perennial ryegrass staggers in grazing animals.     

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.     

Tremorgenic mycotoxins increase gastric smooth muscle activity of sheep reticulum and rumen in vitro

Reticulum and rumen strips (consisting of both muscle layers and the myenteric plexus) were superfused with Tyrode Ringer and their contractions recorded isometrically. The strips were subjected to exogenous acetylcholine and electrical field stimulation (EFS) resulting in contractions that could be blocked by atropine. Responses to the tremorgenic mycotoxin penitrem A and others thought to be involved in ryegrass staggers, paxilline and lolitrem B (10(-10)-10(-6)M), were compared with those of control vehicle (0.1% DMSO). The tremorgens were without effect on quiescent preparations. Penitrem A and paxilline enhanced spontaneously active preparations and the amplitude of contractions in response to EFS. Responses to paxilline had a shorter latency than to penitrem A. Responses of spontaneously active preparations were resistant to atropine. Penitrem A, but not paxilline, increased responses to exogenous acetylcholine. Lolitrem B (10(-6)M) increased responses to EFS, but many responses were equivocal, possibly due to the lower solubility of lolitrem B in aqueous solutions compared to the other tremorgens. The results show that these mycotoxins have peripheral excitatory effects on the reticulorumen and it is suggested that such activity in vivo may reflexly affect centrally derived cyclical contractions.     

Flow-mediated K+ secretion in horses intoxicated with lolitrem B (perennial ryegrass staggers)

AIM: To investigate the effects of lolitrem B intoxication on renal K⁺ secretion in response to increased tubular flow rates.

METHODS: Results are derived from a repeated measure pilot study of seven horses fed non-perennial ryegrass feed for a week prior to exposing them to perennial ryegrass seed and hay that contained an average of 2 ppm lolitrem B. At the end of the control and treatment period frusemide (1 mg/kg I/V) was administered and serial fractional excretion of K⁺(FEK⁺) and fractional excretion of Na⁺(FENa⁺) calculated. Baseline concentration of aldosterone in plasma, serum K⁺concentration and feed K⁺ concentration were also compared.

RESULTS: Key findings included a reduced change in FEK⁺ from 0 to 15 minutes in response to frusemide administration (p=0.022, Wilcoxon signed-rank test) and a reduced baseline concentration of aldosterone in plasma (p=0.022, Wilcoxon signed-rank test) during the treatment period compared with the control.

CONCLUSIONS: Results suggest that lolitrem B intoxication reduced flow-mediated K⁺ secretion and interfered with aldosterone production or secretion. However, further investigation is required to validate these findings and to further elucidate the underlying pathophysiology.

CLINICAL RELEVANCE: Lolitrem B intoxication in horses may cause disruption to electrolyte handling in addition to neurological deficits.     

Ryegrass Staggers: Isolation of Potent Neurotoxins Lolitrem a and Lolitrem B From Staggers-Producing Pastures

Sir, — We wish to report the isolation of two potent neurotoxins from herbage collected from pastures on which the disease of livestock known as ldryegrass staggers rd^(8)(11) occurred. These neurotoxins have been partially characterised by their mass spectral properties (see below) and appear to be new compounds not previously reported. We propose the general name lolitrems for the neurotoxins, based on their association with ryegrass (Lolium perenne L.) and on their ability to produce tremors in animals, and name the isolated compounds lolitrem A and lolitrem B.     

延胡索酸二钠对瘤胃微生物体外发酵不同饲料成分的影响

以黑麦草、高羊茅、稻草、花生壳等粗料成分以及玉米、小麦、可溶性淀粉等精料成分为发酵底物研究了延胡索酸二钠对瘤胃微生物体外发酵的影响。结果表明,延胡索酸二钠显著提高了各种底物的发酵产气量(P<0.01),黑麦草粉、高羊茅草粉、稻草粉和花生壳粉的底物消失率分别比对照组提高了26.22%(P<0.05),14.16%(P<0.05),28.54%(P<0.01)和45.09%(P<0.05);延胡索酸二钠显著提高了小麦、玉米和可溶性淀粉的pH值(P<0.05),但对粗料无明显影响(P>0.05);小麦、玉米和可溶性淀粉处理组的乳酸浓度均低于对照组;各种底物的处理组的NH3-N浓度与对照组无明显差异(P>0.05);各种底物中,小麦的总挥发性脂肪酸(total volatile fatty acid)浓度最高,小麦处理组的乙酸浓度、丙酸浓度、丙酸比例均高于对照组(P<0.05);而乙酸丙酸比降低了0.18(P<0.01),延胡索酸二钠提高了所有底物的丙酸浓度及其比例。综合各指标的发酵情况,延胡索酸二钠对各种底物均有一定的效果,是一种潜在的反刍动物饲料添加剂。     

西藏青稞秸秆与多年生黑麦草混合青贮发酵品质的研究

本研究旨在将青稞秸秆与黑麦草以不同比例混合青贮,评价其发酵品质,筛选出适宜的混合比例。试验设青稞秸秆单独青贮(对照组)、80%青稞秸秆+20%多年生黑麦草混贮(L组)、60%青稞秸秆+40%多年生黑麦草混贮(M组)及40%青稞秸秆+60%多年生黑麦草混贮(H组)4个处理。青贮后第7,14和30天后打开青贮窖,测定青贮饲料发酵品质。结果表明,青稞秸秆与黑麦草混合青贮显著(P<0.05)提高了乳酸和水溶性碳水化合物含量,显著(P<0.05)降低了pH值和干物质含量,M和H组在乳酸产生的同时乙酸含量显著(P<0.05)提高,混贮组氨态氮/总氮略低于对照组(P>0.05),各组均显示较低的丙酸和丁酸含量。从秸秆利用最大化的角度出发,建议以40%黑麦草混合青贮较为适宜。     

瘤胃厌氧真菌对木质素含量不同底物附着及发酵特性研究

结合体外发酵和瘤胃尼龙袋法,研究了本地山羊瘤胃厌氧真菌对木质素含量不同底物(黑麦草叶、黑麦草茎、稻草秸、花生壳,木质素含量依次上升)的附着及发酵特性.结果表明,随体外发酵的进行,真菌数量逐渐增加,以黑麦草叶、黑麦草茎、稻草秸为底物时,瘤胃真菌数量均在48 h达到最大值,且真菌数量的最大值随底物木质素含量的上升呈下降趋势.显微观察显示,体外发酵前72 h附着于稻草秸表面孢子囊数量均显著高于黑麦草叶和黑麦草茎(P<0.05),而切缘孢子囊数量三者差异不显著.瘤胃内培养结果表明,稻草秸切缘和表面孢子囊数量均在48 h时最大,72 h后数量显著下降(P<0.05),而黑麦草茎切缘和表面上的孢子囊数量均在24 h 时最大,72 h后数量显著下降(P<0.05);稻草秸切缘和表面上孢子囊数量均要高于黑麦草茎上的数量.不同底物进行厌氧真菌体外发酵时其累计产气量、干物质消失率和纤维素消失率差异均不显著,但在瘤胃内培养时,各底物的干物质消失率则随底物木质素含量的上升而显著下降(P<0.05).     

Amino acid neurotransmitter release from cerebrocortical synaptosomes of sheep with severe ryegrass staggers in New Zealand

Enhanced unstimulated release of the excitatory amino acid neurotransmitters aspartic acid and glutamic acid was measured in cerebrocortical synaptosomes prepared from sheep showing severe symptoms of ryegrass staggers. A similar pattern of deviation from neurologically normal animals was seen whether the sheep had contracted the disorder naturally or whether they had been deliberately caused to develop the disorder by grazing specific pastures rich in endophyte infected ryegrass. Deranged neurotransmitter release may reasonably be involved in the chemical pathology of ryegrass staggers and the findings are in accord with the putative synonymy of lolitrem toxicosis and ryegrass staggers.     

Effect of Feeding Endophyte-Infected Feed and Bedding on the Performance of Broilers

Two varieties of chopped grass seed straw, tall fescue (Festuca arundinaces L.) and perennial ryegrass (Lolium perenne L.), were identified as alternative sources to wood sawdust or shavings as bedding for use by Pacific Northwest broiler producers. However, some broiler growers expressed concern that straw may be contaminated with endophytes, symbiotic fungi that are known to be toxic to livestock. The primary toxic compound of concern for livestock in endophyte infected grass straw is ergovaline.Two experiments were conducted to determine if broiler performance was affected by either the feeding of endophyte infected grass seed containing ergovaline, or rearing broilers on endophyte infected grass seed straw. The exposure of broilers to either endophyte-infected chopped straw as bedding and/or feeding endophyte infected ground grass seed in the diet with ergovaline levels as high as 423 ppb, did not significantly affect performance or the micro anatomy of kidney or liver tissues.     

Effect of dietary exposure to aflatoxin B1 on resistance of young chickens to organophosphate pesticide challenge

White leghorn chickens were placed on a diet containing 0, 1000, or 3000 ppb aflatoxin B1 for 7 weeks. At the end of that time, the birds were challenged orally with the organophosphate pesticide malathion. A malathion dose of 215 mg/kg resulted in significant clinical signs of cholinergic poisoning in 4/10, 5/10, and 8/10 birds fed 0, 1000, and 3000 ppb aflatoxin B1, respectively, and the chickens required antidotal atropine 40 min later. Activity of brain cholinesterase was significantly lower than control levels in all birds given this dose of malathion, with activities being 28% +/- 6, 21% +/- 2, and 15% +/- 2 of control values (Mean +/- standard error, N = 5) if fed 0, 1000, and 3000 ppb aflatoxin B1, respectively. Plasma cholinesterase values paralleled those of brain, with significantly more inhibition in samples from birds given 1000 and 3000 ppb aflatoxins with malathion. Brain and plasma cholinesterase activities in birds fed aflatoxins and given a dose of malathion below the threshold for cholinergic signs (125 mg/kg) were also lower than activities in birds given malathion alone. Although aflatoxin alone had no direct effect on the activities of these enzymes, it appears that this mycotoxin may contribute to the esterase inhibition that is a manifestation of the acute toxic effects of malathion in chickens.     

Tremorgenic mycotoxins paxilline, penitrem and lolitrem B, the non-tremorgenic 31-epilolitrem B and electromyographic activity of the reticulum and rumen of sheep

The mycotoxic tremorgens penitrem, paxilline and lolitrem B had profound effects on electromyographic (EMG) activity of smooth muscle of the reticulorumen in conscious sheep, with a similar time course of action to their respective characteristic effects on the induction (1 to 2, 15 to 20 and 20 to 30 minutes) and the duration (1 to 2, 1 to 2 and 8 to 12 hours) of tremoring. Responses to penitrem revealed a greater sensitivity of smooth muscle than skeletal muscle. Effects included an inhibition of the vagally-dependent cyclical A and B sequences of contraction of the reticulorumen, an increase in their amplitude and an excitation of local intrinsic activity contributing to elevated baselines and the occurrence of chaotic activity of the reticulum. The excitatory local effects were partially blocked by atropine, indicating that stimulation of muscarinic cholinoceptors was involved. Increased local activity may mediate a reflex inhibition of cyclical contractions. A non-tremorgenic isomer of lolitrem B (31-epilolitrem B) had no effect on the reticulorumen. The intensity and duration of the effects of lolitrem B, up to 12 hours, indicate that severe disruption of digestion may occur in animals grazing endophyte-infected pasture.     

Ryegrass Staggers: The Presence of Lolitrem Neurotoxins in Perennial Ryegrass Seed

Abstract

Extract

Sir:- Recently, two potent neurotoxins named lolitrem A and lolitrem B were isolated from herbage collected from pastures on which the livestock disorder ryegrass staggers (RGS) occurred. et al.⁽¹⁾ Gallagher, R.T., White, E.P. and Mortimer, P.H. 1981. Ryegrass staggers: Isolation of potent neurotoxins lolitrem A and lolitrem B from staggers-producing pastures. N.Z. vet. J., 29: 189–190. [Taylor &; Francis Online], [Web of Science ®] , [Google Scholar] We now wish to report that samples of perennial rygrass (Loliun yeretzrze L.) seed obtained from several locations in New Zealand contained potent neurotoxins which include lolitrem A and lolitrem B. Further, when pellets made from lolitrem neurotoxin-containing seed were fed to sheep, symptoms indistinguishable from those of severe, natural RGS were induced in four sheep, and mild symptoms in three sheep.     

Rhinopneumonia or mycotoxin intoxication? Neurologic phenomena in horses from a riding school

In the course of several days most of the 40 riding-school horses turned out in paddocks developed ataxia of variable severity. Five of these horses showed severe ataxia and tremors, became paralyzed and were euthanized. Eleven privately-owned horses which were stabled on the same premises showed no clinical signs. The most likely diagnosis seemed to be the 'neurological form of EHV1', although the signs were not entirely typical. A few weeks later a second outbreak occurred among the riding-school horses and one of the privately-owned horses also showed signs of ataxia. In the meantime it had been shown that EHV1 titers in paired serum samples had not increased and that the cerebrospinal fluid of one of the severely affected horses was normal. Toxicological examination of hay, delivered just before the first outbreak and stored for the winter, showed a significantly increased concentration of lolitrem B mycotoxin (5-6 mg/kg). The hay appeared to have been made of ryegrass used for lawns and playing fields. Retrospectively it became probable that this hay occasionally been fed to the horses just before the onset of clinical problems. It is concluded that the horses showed the 'ryegrass-stagger syndrome'.     

Mycotoxins as a risk factor for the origin of diseases and production decreases in swine facilities--an epidemiologic study]

Feed samples checked for the mycotoxins zearalenone and ochratoxin A from the harvest 1987 were positive at a markedly higher percentage (37.5%) compared to previous years, which is explained by the especially unfavourable harvesting conditions of 1987. In certain herd problems affecting the digestive or respiratory tract, mycotoxins could be detected with a much higher frequency (64.7% and 50.0% respectively). The mean level detected in feed samples by thin layer chromatography ranged within 30.3 ppb for zearalenone and within 58.3 ppb for ochratoxin A. In most cases there was a history of infertility. Considering the clinical situation, which is presented comparatively in herds with positive mycotoxin results, the possible involvement of mycotoxins in the disease, even at very low concentrations, is pointed out. In this context, zearalenone is incriminated of being an indicator of a multitoxic process besides its own direct effects. According to own experiences low levels of zearalenone in the range of 20-50 ppb in the feed have to be considered hazardous. If changing of pig feed in cases of herd problems will be recommended, a level of less than 10 ppb of zearalenone, especially in sow and piglet rations, should not be exceeded. Same may be valid to ochratoxin A.