Assessment of endophyte-derived tremorgenic compounds in Ipomoea asarifolia using mouse models.

Ipomoea asarifolia has been associated with a tremorgenic syndrome in livestock. Recently indole diterpene compounds were identified in I. asarifolia, some of which have been shown to cause a tremorgenic syndrome. In this study, the tremorgenic nature of I. asarifolia was assessed using a mouse model. Adult mice were fed rodent chow containing 10, 15, 20 and 25% endophyte infected (E+), or 25% endophyte free (E-), I. asarifolia for 14 days. The mice fed E+ chow developed a tremorgenic syndrome as characterized by visually observed muscle tremors and an inability to traverse a balance beam, whereas the mice fed E- chow did not develop tremors and had similar muscle coordination to control mice. A lactating mouse model was also used to determine if the compounds can be transferred to nursing pups via the milk. Nursing pups were exposed via their mother's milk for 21 days, from post-natal day 0-21. The pups from dams exposed to E+ chow developed a similar tremorgenic syndrome. Data presented in this study demonstrate that the tremorgenic compounds in I. asarifolia are endophyte derived. Additionally, both adult mice and nursing pups are good models for studying the tremorgenic nature of I. asarifolia and related plants.

Age-dependent intoxication by larkspur (Delphinium) in Angus steers.

The effect of age on larkspur poisoning of cattle is unknown. An experiment consisting of oral dosing of dried, ground, Delphinium barbeyi to ten Angus steers as yearlings, and again at two years was performed. There was a significant difference between the responses of yearling and two year old steers (P = 0.0015), with yearling steers being more susceptible. These results suggest that the adverse response of Angus cattle to larkspur is age-dependent.

Complete inhibition of fetal movement in the day 40 pregnant goat model by the piperidine alkaloid anabasine but not related alkaloids.

Four chemically similar alkaloids, anabasine, anabaseine, epibatidine and dimethylphenylpiperazinium (DMPP), are potent nicotinic acetylcholine receptor agonists of fetal muscle nicotinic acetylcholine receptors in human TE-671 cells. Based on results with these cells, we hypothesized that the alkaloids would completely inhibit ultrasound-monitored fetal movement in a goat model. Different, single doses of anabasine, anabaseine, epibatidine, DMPP, or saline control were administered I.V. to pregnant goats on day 40 of gestation and the number of fetal movements per 5 min sample was measured by ultrasound at times 0, 0.5, 1, 2, 4 and 8 h. The differences among does in fetal movements were more consistent at dosing and following recovery for doses of anabasine above 0.125 mg/kg compared to the other compounds and dosages. Anabasine actions were dose-dependent with an IC50 value of ∼0.1 mg/kg, and, at a dose of 0.8 mg/kg, completely inhibited fetal movement for 1.5 h after dosing. Anabaseine, epibatidine, and DMPP failed to completely inhibit fetal movement in day 40 pregnant goats at doses predicted to be effective. These results suggest that while experiments with TE-671 cells provide valuable information and predictions of the actions of plant alkaloids on fetal movement, in vivo experiments are still required in order to determine the ability of an alkaloid to inhibit fetal movement in livestock species. Moreover, other pharmacological properties such as receptor differences between mammalian species and differences in the pharmacokinetic properties of the alkaloids also are likely to weaken teratologic predictions based solely on the in vitro data.

Changes in the rumen bacterial microbiome of cattle exposed to ponderosa pine needles.

Consumption of ponderosa pine needles, as well as needles and bark from a number of other trees, can cause abortions in cattle. The abortifacient compounds in these trees are labdane resin acids, including isocupressic acid and agathic acid. Previous research has demonstrated that cattle conditioned to pine needles metabolize the labdane resin acids more quickly than naïve cattle. The results from that study indicated that changes had occurred in the rumen of conditioned cattle. Therefore, in this study, the changes that occurred in the rumen bacterial microflora of cattle during exposure to ponderosa pine needles were evaluated. Cattle were dosed with ground pine needles twice daily for 7 d. Rumen samples were collected on d 0, 3, 7, and 14 (7 d after treatment stopped) and ruminal bacterial microbiome analyses were performed. There were 372 different genera of bacteria identified in the rumen samples. Principal coordinate analysis indicated that there was a significant difference in the rumen bacterial composition between the time points. There were 18 genera that increased in abundance from d 0 to d 7. Twenty three genera decreased in abundance from d 0 to d 7. The results from this study demonstrated that exposure of cattle to pine needles caused a clear shift in the rumen microbiome composition. In general, this shift lasted less than 1 wk post exposure, which indicates that any prophylactic treatment to manipulate the ruminal metabolism of the abortifacient compounds in pine needles would need to be continuously administered to maintain the necessary microbial composition in the rumen.

Serum toxicokinetics after intravenous and oral dosing of larkspur toxins in goats.

Poisoning of cattle by larkspur plants (Delphinium spp.) is a concern for cattle ranchers in western North America. Previous research studies have evaluated the toxicokinetic profile of multiple larkspur toxins in several livestock species. However, those studies were all performed by orally dosing plant material. Consequently some toxicokinetic parameters could not be definitively determined. In this study, we compared the serum toxicokinetic profile of the larkspur alkaloids methyllycaconitine (MLA) and deltaline in goats dosed both IV and via oral gavage. The results from this study indicate that the toxic alkaloids in larkspurs undergo flip-flop kinetics, meaning the rate of absorption of the alkaloids is slower than the rate of elimination. The implications of flip-flop kinetics in treating animals poisoned by larkspur is discussed.

Administering multiple doses of a non N-(methylsuccinimido) anthranoyllycoctonine (MSAL)-containing tall larkspur (Delphinium occidentale) to cattle.

Larkspurs (Delphinium spp.) are a serious toxic plant problem for cattle in western North America. There are two chemotypes of D. occidentale, a more toxic and a less toxic chemotype. The objective of this study was to evaluate the acute toxicity of the less toxic chemotype when administered in multiple doses to cattle. These results suggest that cattle could consume enough of the less toxic chemotype to be poisoned in a range setting.

Comparison of the serum toxicokinetics of larkspur toxins in cattle, sheep and goats.

Larkspurs (Delphinium spp.) are a major cause of cattle losses in western North America, whereas sheep are thought to be resistant to larkspur toxicosis. Goats are often used as a small ruminant model to study poisonous plants. In this study, we compared the serum toxicokinetic profile of toxic larkspur alkaloids from Delphinium barbeyi in cattle, goats, and sheep. The results from this study indicate that kinetic differences could partially explain species differences in susceptibility to larkspur toxicosis.

White snakeroot poisoning in goats: Variations in toxicity with different plant chemotypes.

Tremetone and possibly other benzofuran ketones are believed to be the toxic compounds in white snakeroot. However, disease has not been reproduced with purified toxins and the concentrations of the benzofuran ketones in white snakeroot populations that cause toxicosis have not been documented. The objectives of this study were to compare the toxicity of seven plant populations, better characterize the clinical and pathologic changes of poisoning, and correlate intoxication with benzofuran ketone content. Four of the seven white snakeroot collections were toxic at the dose and duration used in the study. Affected goats became exercise intolerant, had significant serum enzyme changes and histological lesions in the large appendicular muscles. The incidence and severity of poisoning was not correlated with total doses of tremetone or total benzofuran ketone concentrations suggesting they may not be closely involved in producing toxicity and the possible involvement of an unidentified toxin. The results also demonstrate that white snakeroot populations vary chemically and toxicologically.

Elk (Cervus Canadensis) preference for feeds varying in selenium concentration.

Selenium-accumulator plants are reputed to be unpalatable to large ungulates. Elk (Cervus canadensis) populations in southeastern Idaho overlap with populations of Se-rich plants, but there is no information on the influence of plant Se concentration on elk dietary preferences. The objective of this study was to determine, under controlled conditions, the preference of elk for feeds varying in Se concentrations. Seven yearling female elk (128 ± 5 kg) were purchased from a commercial elk farm in southeastern Idaho and adapted to low-Se alfalfa pellets. Three feeding trials using pellets with predetermined Se concentrations were conducted. Alfalfa pellets were commercially made with the addition of Symphyotrichum ascendens (western aster) so that the pellets contained 0.4, 5, 20, 50, or 100 mg/kg (DM basis) Se. In trial 1, 5 Se-containing alfalfa pellets (0.4, 5, 20, 50, and 100 mg/kg Se) were offered for 10 d; trial 2 used 4 Se-containing alfalfa pellet choices (0.4, 20, 50, and 100 mg/kg), and trial 3 used 3 pellet choices (0.4, 50, and 100 mg/kg) for 6 d. In trial 1, consumption of the control pellets by elk was greater than each of the other pellet choices (P < 0.001). Similarly, consumption of the 5-mg/kg Se pellet differed from control pellet and all other Se-containing pellets (P < 0.0001). There were no differences (P > 0.50) in consumption of the 20-, 50-, or 100-mg/kg Se pellets. In trial 2, elk consumed more (P < 0.0001) of the control pellet than the 20-, 50-, and 100-mg/kg Se pellets. Similarly, elk consumed more (P < 0.0001) of the 20-mg/kg Se pellet than the 50- and 100-mg/kg Se pellets. There were no differences (P > 0.99) in elk consumption of the 50- and 100-mg/kg Se pellets. In trial 3, elk consumption of the control and 50- and 100-mg/kg Se pellets differed (P ≤ 0.03) from one another each day except that on d 1 and 2, where elk consumption of the 50- and 100-mg/kg Se pellets did not differ (P ≥ 0.32). Elk clearly discriminated against pellets with the highest Se concentrations when they were given pellets with differing Se concentrations. These results suggest that elk are not likely to select forages with high Se concentrations, particularly when high-Se plants are present in a rangeland situation with numerous other forage choices.

The effect of administering multiple doses of tall larkspur (Delphinium barbeyi) to cattle.

Larkspurs ( spp.) are one of the most serious toxic plant problems on foothill and mountain rangelands in the western United States. A considerable amount of research has been conducted over the years in both field and pen settings. The results of these research efforts have significantly increased our understanding of the poisoning of cattle by larkspurs. However, most of the pen studies conducted thus far have used a dosing regimen of a single bolus dose, which does not accurately mimic the manner by which cattle are poisoned by larkspur while grazing. Consequently, the objective of this study was to evaluate the acute toxicity of tall larkspur ( collected near Manti, UT) when administered in multiple doses, with the intent to identify a no observable adverse effect level (NOAEL). The adverse effect selected for this study was muscle weakness to the point the cattle could no longer remain ambulatory as would be required in a grazing environment, thus becoming sternally recumbent when exercised. Hereford steers were administered various doses of tall larkspur at 12-h intervals for 4 d or until they showed marked signs of muscle weakness. The results suggest that a dose of 2 mg kg∙d -(methylsuccinimido) anthranoyllycoctonine (MSAL)-type alkaloids is the NOAEL for a tall larkspur population with a norditerpenoid alkaloid profile containing 4 mg MSAL-type alkaloids/g plant material and 12 mg non-MSAL-type alkaloids/g plant material. Additionally, a computer model was generated to simulate multiple-dosing regimens at the various doses as well as different dosing regimens. The results from this study suggest that a 500-kg steer can consume a daily dose of 1.25 kg of fresh tall larkspur (with a similar alkaloid profile) without becoming severely poisoned (suffering from muscle weakness to the point of recumbency). Additionally, these results indicate that a serum concentration of approximately 355 ng methyllycaconitine/mL may represent a toxic threshold.

Studies on the teratogenicity of anabasine in a rat model.

A number of plant toxins have been shown to be teratogenic to livestock. The teratogenic action of some of these alkaloids is mediated by nicotinic acetylcholine receptors (nAChR). However, for many of these alkaloids it is difficult to obtain sufficient quantities of individual alkaloids to perform teratology studies in livestock species. Therefore the objective of this study was to determine if a rat model can be utilized to characterize the teratogenic nature of individual plant toxins that are nAChR agonists. In this study, we evaluated the teratogenicity of anabasine by feeding pregnant rats anabasine-containing rodent chow from gestational day (GD) 6-21. On GD21, the dams were euthanized and the gravid uteri were removed. The gravid uteri and individual pups were weighed. The pups were evaluated for bone malformations including cleft palate and scoliosis. Overall, the results of this study suggest that the rat is not a good model to study the teratogenicity of plant toxins that are nAChR agonists. It is possible that in the rat model, anabasine administered orally via the chow may not result in sufficient reduction in fetal movement to cause the significant malformations observed in livestock species.

Effect of selenium concentration on feed preferences by cattle and sheep.

Selenium-accumulator plants are reputed to be unpalatable to livestock. The objective of this study was to determine if sheep and cattle could discriminate between forages and feeds with different concentrations of Se. In the first study, cattle and sheep preferences for intermediate wheatgrass (Thinopyrum intermedium), alfalfa (Medicago sativa), and western aster (Symphyotrichum ascendens) of varying Se concentrations were assessed. The Se concentrations ranged from 0.8 to 50 mg/kg (DM) in grass, 1.4 to 275 mg/kg in alfalfa, and 4 to 4,455 mg/kg in aster. Selenium concentration had no influence (P > 0.05) on the initial or subsequent preferences of sheep or cattle for grass or alfalfa. Cattle developed an aversion to aster after consuming 95% of the plant material during the first brief exposure and subsequently refused to eat any aster. Sheep consumption of aster was variable, but their preference was not driven by Se concentration. In the next study, cattle and sheep were offered pellets at 1.5% of BW (as fed) that contained increasing concentrations of Se from aster (control and 5, 25, 45, and 110 mg/kg Se). In trial 1, all pellets were offered. In Trials 2 and 3, all pellets were offered with the exception of the 5 mg/kg Se pellet and the 5 and 25 mg/kg Se pellets, respectively. In trial 1, consumption of the control pellet by cattle was greater on all days compared with other Se pellets (P < 0.001). Cattle ate more (P < 0.001) of the 5 mg/kg Se pellet than the higher Se pellets on d 3, 4, and 5. Sheep ate greater amounts of the control and 5 and 110 mg/kg Se pellets compared with the 25 and 45 mg/kg Se pellets (P < 0.0001) on d 1, and sheep consumed primarily the control and 5 mg/kg Se pellets thereafter. In trial 2, cattle and sheep consumed more (P < 0.0001) of the control Se pellet than the 25, 45, and 110 mg/kg Se pellets. In trial 3, cattle consumption of the control and 45 and 110 mg/kg Se pellets differed on d 2 and 3 (P < 0.001), except there was no difference (P > 0.95) in cattle consumption of the control and 45 mg/kg Se pellets on d 1. Sheep consumed primarily the control and 45 mg/kg Se pellets. We conclude that high Se concentrations in fresh forages had no effect on initial consumption by cattle or sheep. When given Se pellets, initial responses were variable, but the results indicate that cattle and sheep adjusted their intake over time to avoid excessive intake of Se.

The effect of low larkspur (Delphinium spp.) co-administration on the acute toxicity of death camas (Zigadenus spp.) in sheep.

In most cases where livestock are poisoned by plants in a range setting, there is more than one potential poisonous plant in the same area. Two poisonous plants that are often found growing simultaneously in the same location are death camas (Zigadenus spp.) and low larkspur (Delphinium spp.). Sheep are known to be susceptible to death camas poisoning while they are thought to be resistant to larkspur. The objective of this study was to determine if co-administration of low larkspur would exacerbate the toxicity of death camas in sheep. A dose finding study was performed to find a dose of death camas that caused minimal clinical signs of poisoning. Sheep were observed for clinical signs of poisoning as well as changes in heart rate and muscle fatigue. Sheep dosed with 1.14 g of death camas per kg BW showed slight frothing and lethargy, whereas sheep dosed with death camas and low larkspur showed slightly more noticeable clinical signs of poisoning. Sheep dosed with only low larkspur, at 7.8 g/kg BW, showed no signs of poisoning. Although we observed a qualitative difference in clinical signs of intoxication in sheep co-treated with death camas and low larkspur we did not detect any quantitative differences in heart rate, exercise-induced muscle fatigue, or differences in serum zygacine kinetics. Consequently, the results from this study suggest that low larkspur does not affect the toxicity of death camas in sheep. The results from this study increase knowledge and understanding regarding the acute toxicity of death camas and low larkspur in sheep. As combined intoxications are most likely common, this information will be useful in further developing management recommendations for ranchers and in designing additional experiments to study the toxicity of death camas to other livestock species.

Comparative oral dose toxicokinetics of selenium compounds commonly found in selenium accumulator plants.

Consumption of Se accumulator plants by livestock can result in Se intoxication. Recent research indicates that the Se forms most common in Se accumulator plants are selenate and Se-methylselenocysteine (MeSeCys). In this study the absorption, distribution, and elimination kinetics of Se in serum and whole blood of lambs dosed with a single oral dose of (1, 2, 3, or 4 mg Se/kg BW) of sodium selenate or MeSeCys were determined. The Se concentrations in serum and whole blood for both chemical forms of Se followed simple dose-dependent relationships. Se-methylselenocysteine was absorbed more quickly and to a greater extent in whole blood than sodium selenate, as observed by a greater peak Se concentration (Cmax; P < 0.0001), and faster time to peak concentration (Tmax; P < 0.0001) and rate of absorption (P < 0.0001). The rate of absorption and Tmax were also faster (P < 0.0001) in serum of lambs dosed with MeSeCys compared with those dosed sodium selenate at equimolar doses; however, Cmax in serum was greater (P < 0.0001) in lambs dosed with sodium selenate compared with those dosed MeSeCys at equimolar doses. The MeSeCys was absorbed 4 to 5 times faster into serum and 9 to 14 times faster into whole blood at equimolar Se doses. There were dose-dependent increases in the area under the curve (AUC) for Se in serum and whole blood of lambs dosed with both sodium selenate and MeSeCys. In whole blood the MeSeCys was approximately twice as bioavailable as sodium selenate at equimolar doses as observed by the AUC, whereas in serum there were no differences (P > 0.05) in AUC at the same doses. At 168 h postdosing the Se concentration in whole blood remained much greater (P < 0.0001) in lambs dosed with MeSeCys as compared with lambs dosed with sodium selenate; however, the serum Se concentrations were not different between treatments at the same time point. The results presented in this study demonstrate that there are differences between the kinetics of different selenocompounds when orally dosed to sheep. Therefore, in cases of acute selenosis, it is important to understand the chemical form to which an intoxicated animal was exposed when determining the importance and meaning of Se concentration in serum or whole blood obtained at various times postexposure.

The role of the α7 subunit of the nicotinic acetylcholine receptor on motor coordination in mice treated with methyllycaconitine and anabasine.

The adverse effects of methyllycaconitine (MLA) have been attributed to competitive antagonism of nicotinic acetylcholine receptors (nAChR). Research has indicated a correlation between the LD50 of MLA and the amount of α7 nAChR in various mouse strains, suggesting that mice with more α7 nAChR require more MLA to be poisoned. However, recent research demonstrated that there was no difference in the acute lethality (LD50 ) to MLA in mice lacking the α7 nAChR subunit compared with wild-type mice. The objective of this study was to determine if the α7 nAChR subunit plays a role in motor coordination deficiencies that result from exposure to nAChR antagonists and agonists. We compared the motor function and coordination in wild-type mice to mice lacking the α7 subunit of the nAChR, after treating them with a non-lethal dose of MLA or anabasine, using the following tests: balance beam, grip strength, rotarod, open field and tremor monitor. Analysis of the data indicated that overall there was no difference between the wild-type and knockout mice (P = 0.39 for grip strength; P = 0.21 for rotarod; P = 0.41 for balance beam; P = 0.22 for open field; and P = 0.62 for tremors). Thus results from this study suggest that α7 nAChR does not play an integral role in the acute effects of MLA or anabasine on motor function/coordination. Consequently other subunits of nAChRs found in the neuromuscular junction are likely the primary target for MLA and anabasine resulting in motor coordination deficiencies and acute toxicosis.

The role of the α7 subunit of the nicotinic acetylcholine receptor in the acute toxicosis of methyllycaconitine in mice.

The adverse physiological effects of methyllycaconitine (MLA) have been attributed to its competitive antagonism of nicotinic acetylcholine receptors (nAChRs). Recent research suggested a correlation between the lethal dose (LD50 ) of MLA and the amount of α7 nAChR in various mouse strains, suggesting that mice with more α7 nAChR require more MLA to be poisoned. The objective of this study was to characterize the role of the α7 subunit in the acute toxicosis of MLA by evaluating the acute toxicity of MLA in mice lacking the α7 subunit. The LD50 values for MLA were 4.2 ± 0.9, 3.7 ± 1.1 and 3.3 ± 0.9 mg kg(-1) body weight (BW) for wild-type, heterozygous knockout and homozygous knockout mice, respectively. We also evaluated the response of anabasine in these mice. The LD50 values for anabasine were 1.6 ± 0.3, 2.0 ± 0.4 and 1.8 ± 0.3 mg kg(-1) BW for wild-type, heterozygous knockout and homozygous knockout mice, respectively. The protein expresson of various nAChR subunits was compared to determine if mice lacking the α7 subunit compensate by over expressing other nAChR subunits. There were no significant differences in the protein expression of the α3 , α4 , α5 , ß2 and ß4 subunits amongst the three genotypes of mice in brain or skeletal muscle. The results of this study suggest that α7 nAChR does not play an integral role in the acute toxicosis of MLA or anabasine. Consequently other nAChR subunits of nAChRs found in the neuromuscular junction are probably the primary target for MLA and anabasine resulting in acute toxicosis.

A comparison of the metabolism of the abortifacient compounds from Ponderosa pine needles in conditioned versus naive cattle.

Isocupressic acid (ICA) is the abortifacient compound in ponderosa pine (Pinus ponderosa L.) needles, which can cause late-term abortions in cattle (Bos taurus). However, cattle rapidly metabolize ICA to agathic acid (AGA) and subsequent metabolites. When pine needles are dosed orally to cattle, no ICA is detected in their serum, whereas AGA is readily detected. Recent research has demonstrated that AGA is also an abortifacient compound in cattle. The observation has been made that when cattle are dosed with labdane acids for an extended time, the concentration of AGA in serum increases for 1 to 2 d but then decreases to baseline after 5 to 6 d even though they are still being dosed twice daily. Therefore, in this study we investigated whether cattle conditioned to pine needles metabolize ICA, and its metabolites, faster than naïve cattle. Agathic acid was readily detected in the serum of naïve cattle fed ponderosa pine needles, whereas very little AGA was detected in the serum of cattle conditioned to pine needles. We also compared the metabolism of ICA in vitro using rumen cultures from pine-needle-conditioned and naïve cattle. In the rumen cultures from conditioned cattle, AGA concentrations were dramatically less than rumen cultures from naïve cattle. Thus, an adaptation occurs to cattle conditioned to pine needles such that the metabolism AGA by the rumen microflora is altered.

The effect of 7, 8-methylenedioxylycoctonine-type diterpenoid alkaloids on the toxicity of tall larkspur (Delphinium spp.) in cattle.

Delphinium spp. contain numerous norditerpenoid alkaloids which are structurally delineated as 7, 8-methylenedioxylycoctonine (MDL) and N-(methylsuccinimido) anthranoyllycoctonine (MSAL)-type alkaloids. The toxicity of many tall larkspur species has been primarily attributed to their increased concentration of MSAL-type alkaloids, such as methyllycaconitine (MLA), which are typically 20 times more toxic than MDL-type alkaloids. However, the less toxic MDL-type alkaloids are often more abundant than MSAL-type alkaloids in most Delphinium barbeyi and Delphinium occidentale populations. Previous research demonstrated that MDL-type alkaloids increase the acute toxicity of MSAL-type alkaloids. In this study, we examined the role of MDL-type alkaloids on the overall toxicity of tall larkspur plants to cattle while controlling for the exact dose of MSAL-type alkaloids. Cattle were dosed with plant material from 2 different populations of tall larkspur containing either almost exclusively MDL- or MSAL-type alkaloids. These 2 plant populations were combined to create mixtures with ratios of 0.3:1, 1:1, 5:1, and 10:1 MDL- to MSAL-type alkaloids. The dose that elicited similar clinical signs of poisoning in mice and cattle was determined for each mixture on the basis of the MSAL-type alkaloid content. As the ratio of MDL- to MSAL-type alkaloids increased, the amount of MSAL-type alkaloids required to elicit clinical signs decreased. These results indicate that the less toxic MDL-type alkaloids in tall larkspur exacerbate the toxicity of the MSAL-type alkaloids. Consequently, both the amount of MSAL-type alkaloids and the amount of total alkaloids should be fully characterized to determine more accurately the relative toxicity of tall larkspur plant material.

The acute toxicity of the death camas (Zigadenus species) alkaloid zygacine in mice, including the effect of methyllycaconitine coadministration on zygacine toxicity.

Death camas (Zigadenus spp.) is a common poisonous plant on foothill rangelands in western North America. The steroidal alkaloid zygacine is believed to be the primary toxic component in death camas. Poisonings on rangelands generally occur in the spring when death camas is abundant, whereas other more desirable forage species are limited in availability. In most cases where livestock are poisoned by plants in a range setting, there is more than one potential poisonous plant in that area. One common poisonous plant that is often found growing simultaneously in the same area as death camas is low larkspur (Delphinium nuttallianum). Consequently, the objectives of this study were to conduct acute toxicity studies in mice and to determine if coadministration of low larkspur will exacerbate the toxicity of death camas. We first characterized the acute toxicity of zygacine in mice. The LD(50) of zygacine administered intravenously (i.v.) and orally was 2.0 ± 0.2 and 132 ± 21 mg/kg, respectively. The rate of elimination of zygacine from whole blood was determined to be 0.06 ± 0.01/min, which corresponds to an elimination half-life of 13.0 ± 2.7 min. The i.v. LD(50) of total alkaloid extracts from a Utah and a Nevada collection were 2.8 ± 0.8 and 2.2 ± 0.3 mg/kg, respectively. The i.v. LD(50) of methyllycaconitine (MLA), a major toxic alkaloid in low larkspur, was 4.6 ± 0.5 mg/kg, whereas the i.v. LD(50) of a 1:1 mixture of MLA and zygacine was 2.9 ± 0.7 mg/kg. The clinical signs in mice treated with this mixture were very similar to those of mice treated with zygacine alone, including the time of onset and death. These results suggest that there is an additive effect of coadministering these 2 alkaloids i.v. in mice. The results from this study increase knowledge and understanding regarding the acute toxicity of death camas. As combined intoxications are most likely common, this information will be useful in further developing management recommendations for ranchers and in designing additional experiments to study the toxicity of death camas to livestock.

Neem-tree (Azadirachta indica Juss.) extract as a feed additive against the American dog tick (Dermacentor variabilis) in sheep (Ovis aries).

Acaricides can be conveyed to ticks via the blood of their hosts. As fruit and kernel extracts from the Meliaceae family, and, in particular the tetranortriterpenoid azadirachtin (AZA) inhibits tick egg production and embryogenesis in the Ixodidae ticks, we investigated the effects of Neem Azal, an extract containing 43% AZA, given as a feed additive to lambs artificially infested with engorging adult Dermacentor vairiabilis ticks. After tick attachment, the lambs were allotted to three dietary treatments: AZA0 (control, n=10), AZA0.3 (n=5), and AZA0.6 (n=5), with feed containing 0%, 0.3%, and 0.6% AZA on DM basis, respectively. In half of the AZA0 lambs, ticks were sprayed on day 4 after attachment with an ethanol:water:soap emulsion containing 0.6% AZA (AZA0S). In spite of its very pungent odor, the neem extract was well accepted by all but one lamb. No differences were found between treatment groups in liver enzymes in blood, and there was no indication of toxicity. The plasma AZA concentrations after 7 and 14 days of feeding AZA were (4.81 and 4.35 microg/mL) for the AZA0.6 and (3.32 and 1.88 microg/mL) for the AZA0.3 treatments, respectively (P<0.0001). Treatments were not lethal to ticks, but tick weights at detachment were 0.64, 0.56, 0.48, and 0.37 g for ticks from the AZA0, AZA0.3, AZA0S, and AZA0.6 treatments (P<0.04), respectively, suggesting that blood AZA impaired blood-feeding. The highest mortality rate after detachment was for AZA0.6 (P<0.09). As AZA affects embryo development and ticks at the molting stages, we expect that following treatments of hosts for longer periods, one-host ticks will be more affected than the three-host tick D. variabilis.