Acute death as a result of poisoning tropical (Bos taurus indicus) but not temperate (Bos taurus taurus) cattle after oral dosing with Lupinus leucophyllus (velvet lupine).

Due to climate change and increasing summer temperatures, tropical cattle may graze where temperate cattle have grazed, exposing tropical cattle to toxic plants they may be unfamiliar with. This work compared the toxicity of Lupinus leucophyllus (velvet lupine) in temperate and tropical cattle. Orally dosed velvet lupine in tropical cattle caused death. If producers opt to graze tropical cattle, additional care must be taken on rangelands where toxic lupines like velvet lupine grow.

Ruminant metabolism of zygacine, the major toxic alkaloid in foothill death camas (Zigadenuspaniculatus).

Death Camas (Zigadenus spp.) are common poisonous plants distributed throughout North America. The toxic alkaloids in foothill death camas are zygadenine and a series of zygadenine esters, with zygacine, the 3-acetyl ester of zygadenine, being the most abundant. Both cattle and sheep can be poisoned by grazing death camas, however, sheep consume death camas more readily and are most often poisoned. We hypothesized that the presence of enzymes, including esterases present in the rumen, liver, and blood of livestock would metabolize zygacine. The objective of this study was to investigate the metabolism of zygacine in sheep and cattle using in-vitro and in-vivo systems. Results from experiments where zygacine was incubated in rumen culture, plasma, liver S9 fractions, and liver microsomes and from the analysis of rumen and sera from sheep and cattle dosed death camas plant material demonstrated that zygacine is metabolized to zygadenine in the rumen, liver and blood of sheep and cattle. The results from this study indicate that diagnosticians should analyze for zygadenine, and not zygacine, in the rumen and sera for the diagnosis of livestock suspected to have been poisoned by foothill death camas.

Biomarkers and their potential for detecting livestock plant poisonings in Western North America.

The United States National Cancer Institute defines a biomarker as: "A biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or disease." In Veterinary Medicine, biomarkers associated with plant poisonings of livestock have great utility. Since grazing livestock poisoned by toxic plants are often found dead, biomarkers of plant poisoning allow for a more rapid postmortem diagnosis and response to prevent further deaths. The presence and concentration of toxins in poisonous plants are biomarkers of risk for livestock poisoning that can be measured by the chemical analysis of plant material. More difficult is, the detection of plant toxins or biomarkers in biological samples from intoxicated or deceased animals. The purpose of this article is to review potential biomarkers of plant poisoning in grazing livestock in the Western North America including recently investigated non-invasive sampling techniques. Plants discussed include larkspur, lupine, water hemlock, swainsonine-containing plants, selenium-containing plants, and pyrrolizidine alkaloid containing plants. Other factors such as animal age and sex that affect plant biomarker concentrations in vivo are also discussed.

Evaluation of diazepam as a drug treatment for water hemlock (Cicuta species) poisoning in Spanish goats.

Water hemlocks (Cicuta spp.) are toxic members of the Apiaceae plant family. The best drug treatment for the convulsions associated with acute water hemlock poisoning in livestock and humans has not been determined experimentally. This work compared the therapeutic actions of benzodiazepines (diazepam) and barbiturates (phenobarbital) on water hemlock poisoning in a goat model. C. maculata tubers were orally dosed to goats. Experimental groups consisted of; control saline; 20 mg/kg phenobarbital; 1.0 mg/kg diazepam; 10 mg/kg diazepam; and 1.0 mg/kg diazepam administered as needed to moderate convulsions by intravenous (i.v.) infusion. Diazepam provided nearly instant control of convulsions. Clinical signs of poisoning were completely controlled for the duration of the experiment in the goats that received the 10 mg/kg diazepam dose. These results suggest that diazepam is effective at managing the clinical signs of water hemlock poisoning in goats. We speculate that diazepam can be used as a potential treatment for water hemlock poisoning in other livestock species and humans.

Microsomal activation, and SH-SY5Y cell toxicity studies of tremetone and 6-hydroxytremetone isolated from rayless goldenrod (Isocoma pluriflora) and white snakeroot (Agertina altissima), respectively.

This research compared the cytotoxic actions of the benzofuran ketone, tremetone in B16 murine melanoma cells to SH-SY5Y human neuroblastoma cells with an MTT assay. Tremetone was not cytotoxic in B16 cells. In SH-SY5Y cells, concentration-dependent tremetone cytotoxicity occurred without microsomal activation. No cytotoxicity was observed with 6-hydroxytremetone. This suggests that SH-SY5Y cells are a better model for the cytotoxic actions of tremetone and that tremetone is toxic without microsomal activation.

The comparative toxicity of Isocoma species in calves.

Isocoma pluriflora and Isocoma acradenia are toxic plants that contain the putative toxin tremetone. It is common for I. pluriflora to poison livestock in the southwestern United States. I. acradenia has been suspected of poisoning livestock but its toxicity has not been confirmed by association with clinical poisonings or experimental studies. Jersey calves dosed with I. pluriflora and I. acradenia for nine days developed "trembles" characterized by skeletal muscle degeneration and necrosis and large increases in serum creatine kinase activity. This is the first report of I. acradenia toxicity in an animal model. This study also demonstrates that I. pluriflora remains toxic even though tremetone concentrations in the plant were low due to storage of the plant for over five years. Thus, supporting recent research which indicates that another toxin in the plant may be responsible for, or at least contributes to causing "trembles" in livestock.

Investigation of the Possible Pharmacologically Active Forms of the Nicotinic Acetylcholine Receptor Agonist Anabaseine.

Three major forms of the nicotinic agonist toxin anabaseine (cyclic iminium, cyclic imine and the monocationic open-chain ammonium-ketone) co-exist in almost equal concentrations at physiological pH. We asked the question: Which of these forms is pharmacologically active? First, we investigated the pH dependence of anabaseine inhibition of [3H]-methylcarbamylcholine binding at rat brain α4ß2 nicotinic acetylcholine receptors (nAChRs). These experiments indicated that one or both monocationic forms interact with the orthosteric binding site for ACh. However, since they occur at equal concentrations near physiological pH, we employed another approach, preparing a stable analog of each form and examining its agonist activities and binding affinities at several vertebrate brain and neuromuscular nAChRs. Only 2-(3-pyridyl)-1,4,5,6-tetrahydropyrimidine monohydrogen chloride (PTHP), the cyclic iminium analog, displayed nAChR potencies and binding affinities similar to anabaseine. The cyclic imine analog 2,3'-bipyridyl and the open-chain ammonium-ketone analog 5-methylamino-1-(3-pyridyl)-1-pentanone (MAPP), displayed ≤1% of the activity predicted if the one form was solely active. The lower potency of weakly basic 2,3'-bipyridyl can be explained by the presence of a small concentration of its monocationic form. Since the open chain ammonium-ketone monocationic form of anabaseine has some structural similarity to the neurotransmitter GABA, we also tested the ability of anabaseine and its 1,2-dehydropyrrolidinyl analog myosmine to activate a mammalian GABAA receptor, but no activity was detected. We conclude that the monocationic cyclic iminium is the form which avidly binds and activates vertebrate nAChRs.

Animal and plant factors which affect larkspur toxicosis in cattle: Sex, age, breed, and plant chemotype.

Larkspur (Delphinium spp.) poisoning is a long-term problem for cattle grazing on rangelands of western North America. Recent research has shown that both plant and animal-based factors are critical in understanding and mitigating larkspur poisoning in cattle. Non-toxicological factors including sex, age, cattle breed, and plant chemotype affect cattle responses to larkspur. For example, Angus heifers are more susceptible to larkspur intoxication than are steers or bulls. Young cattle appear to be more susceptible to larkspur poisoning than mature animals. Beef breeds of cattle are more susceptible to larkspur intoxication than dairy breeds. In addition to animal factors, plant alkaloid composition (chemotype) affects the potential toxicity for cattle because of differences in the ratios and concentrations of highly toxic N-(methylsuccinimido) anthranoyllycoctonine (MSAL)-type alkaloids compared to less lethal non-MSAL-type alkaloids. Animal- and plant-based factors can provide substantial information to inform livestock producers on management to reduce risk and cattle losses to various larkspur species in western North America.

Evaluation of noninvasive specimens to diagnose livestock exposure to toxic larkspur (Delphinium spp.).

Larkspurs (Delphinium spp.) are native perennial plants that have a serious toxic potential to cattle on foothill and mountain rangelands in the western United States. Livestock death due to larkspur toxicity is attributed to norditerpenoid alkaloids. Diagnosing plant poisonings in livestock is often challenging. The objective of this study was to evaluate the use of three matrices; earwax, oral fluid, and nasal mucus, as noninvasive specimens to determine livestock exposure to larkspurs. Reversed phase high performance liquid chromatography - high resolution mass spectrometry was used to analyze for norditerpene alkaloids, in all three matrices, in cattle administered a single dose of larkspur. Earwax, oral fluid, and nasal mucus were collected over 6 days post-dosing. Methyllycaconitine (MLA) and deltaline concentrations in earwax ranged from 0.4 ±â€¯0.1 to 0.2 ±â€¯0.06 and 0.6 ±â€¯0.5 to 0.11 ±â€¯0.08 ng/mg, respectively. MLA and deltaline concentrations in oral fluid ranged from 0.08 ±â€¯0.03 to 0.01 ±â€¯0.002 ng/mg and 0.07 ±â€¯0.03 ng/mg to not detected (ND), respectively. MLA and deltaline concentrations in nasal mucus ranged from 0.2 ±â€¯0.13 to 0.03 ±â€¯0.01 ng/mg and 0.2 ±â€¯0.12 ng/mg to ND, respectively. The ability to detect differing norditerpene alkaloid chemotypes from two different Delphinium spp. was also possible in the three matrices. This study demonstrates the potential of earwax, oral fluid, and nasal mucus as noninvasive specimens for chemical analyses to aid in the diagnosis of livestock that may have been exposed to and poisoned by larkspur plants.

The effect of alkaloid composition of larkspur (Delphinium) species on the intoxication of Angus heifers1.

Cattle losses from larkspur (Delphinium spp.) toxicity are a long-term challenge on the rangelands of western North America. In addition to animal factors that affect livestock poisonings, plant alkaloid composition (chemotype) affects the intoxication of cattle because some chemotypes are significantly more toxic. Differences in larkspur chemotype toxicity are due to the ratios of N-(methylsuccinimido) anthranoyllycoctonine (MSAL)-type alkaloids to non-MSAL-type alkaloids and the concentrations of those alkaloids in the plant material. The objective of this study was to compare the responses of 6 Angus heifers to 6 chemotypes of larkspur using a Latin square study design. These Angus heifers from the USDA-ARS, Meat Animal Research Center in Clay Center, NE, were chosen for this research because they are the most larkspur-susceptible cattle observed to date. The 6 heifers were given an oral dose of dried ground larkspur and tested for muscle weakness with an exercise test (i.e., walk time). The 6 chemotypes of larkspur had non-MSAL to MSAL-type alkaloid ratios ranging from 1.4:1 to 6:1 and were administered at an oral dose of 7.5 mg/kg MSAL-type alkaloids BW. There was a treatment effect due to larkspur chemotype (P < 0.0001), and period effects were not significant (P = 0.6). There were also significant correlations between the length of time walking on a dirt track at 5 to 6 km/h, and total alkaloid dose (r = -0.92, P = 0.0045) and alkaloid ratio (r = -0.81, P = 0.0258). Serum alkaloid concentrations at 24 h after dosing were representative of the relative abundance of the alkaloid in the plant material. Results from this work suggest that total alkaloid concentrations in combination with alkaloid ratios can be used together to accurately predict the plant risk component of larkspur poisoning to grazing cattle. Animal factors such as cattle age, breed, and sex must also be considered to comprehensively manage larkspur risk.

Sex-dependent differences for larkspur (Delphinium barbeyi) toxicosis in yearling Angus cattle1.

Larkspur (Delphinium spp.) poisoning is a long-term problem for cattle grazing on rangelands of western North America. Results from preliminary experiments have suggested that differences in larkspur toxicity may exist between heifers and bulls. The objective of this study was to compare the physiological responses of yearling Angus heifers, steers, and bulls with a standardized dose of Delphinium barbeyi and to test the hypothesis that the response is sex dependent. Clinical signs of intoxication, including muscle coordination and function, were measured 24 h after oral dosing with larkspur by walking the cattle at a pace of 5 to 6 km h-1 for up to 40 min on an oval dirt track. Due to the experimental methods used, the variation in susceptibility to larkspur was not quantifiable for walking times of 0 or 40 min or more. Larkspur susceptible animals that were not able to walk (0 min; 36% of the animals) or larkspur resistant animals that walked the entire test period of 40 min (9% of the animals) resulted in censored or truncated data. The statistical methods (censReg and lmec) were used to adjust for data truncation or censoring. The heifers were only able to walk -8.9 ± 3.9 min (65.5% censored on the left) compared with 13.2 ± 3.7 min for bulls and 15.9 ± 2.7 min for steers. When heifers were compared with bulls and steers together, heifers walked 23.4 ± 4.5 min less (P < 0.0001). Serum alkaloid concentrations were measured immediately before walking, and deltaline concentrations averaged 266 ± 28, 131 ± 20, and 219 ± 28 ng mL-1 for all heifers, steers, and bulls, respectively, and serum methyllycaconitine concentrations averaged 660 ± 46, 397 ± 32, and 612 ± 34 ng mL-1 for all heifers, steers, and bulls, respectively. The relative risk of a zero walk time for yearling heifers is 330% that of yearling bulls (P = 0.0008). These results suggest that yearling Angus heifers are more susceptible to larkspur intoxication and, when possible, heifers should be kept from grazing larkspur-infested rangelands as a simple management tool to reduce the risk of fatal poisoning.

An Evaluation of Hair, Oral Fluid, Earwax, and Nasal Mucus as Noninvasive Specimens to Determine Livestock Exposure to Teratogenic Lupine Species.

The livestock industry in the western United States loses an estimated $500 million annually from livestock production losses due to poisonous plants. Poisoning of livestock by plants often goes undiagnosed because there is a lack of appropriate or available specimens for analysis. The Lupinus species represent an important toxic plant in western North America that can be toxic and/or teratogenic to livestock species due to the quinolizidine alkaloids. The objective of this study was to evaluate the potential of using earwax, hair, oral fluid, and nasal mucus as noninvasive specimens to determine livestock exposure to the teratogenic Lupinus species. Quinolizidine alkaloids were detected in these four matrices in cattle that were administered a single dose of Lupinus leucophyllus. In addition, quinolizidine alkaloids from lupine were detected in the earwax of cattle that grazed on lupine-infested rangelands. This study demonstrates the potential of earwax, hair, oral fluid, and nasal mucus as noninvasive specimens for chemical analyses to aid in the diagnosis of livestock that may have been exposed to and poisoned by plants.

Effect of grinding and long-term storage on the toxicity of white snakeroot (Ageratina altissima) in goats.

White snakeroot (Ageratina altissima) contains the putative toxin tremetone and can produce a disease called "trembles" or "milk sickness". However the toxicity of tremetone has not been demonstrated in vivo. It has been reported that the plant is less toxic after drying and grinding. The objectives of these studies were to determine: 1) the toxic effect of grinding white snakeroot 4 months prior to dosing and, 2) the toxic effect of storing white snakeroot at ambient temperature for 5 years. Dried white snakeroot, ground 1 day, 1 month, and 4 months prior to dosing, was orally gavaged to goats at 2% of their body weight for up to 28 days or until they were minimally poisoned (minimal muscular weakness and increased serum creatine kinase (CK) activities). All four goats dosed with white snakeroot that had been ground 4 months previously and stored at room temperature were poisoned, became exercise intolerant, and had increased serum CK activities (>5600 U/ L). White snakeroot stored for 5 years was toxic as 3 of 5 dosed goats developed clinical disease within only 6 days of dosing even though approximately 80% of the tremetone in the plant had disappeared during the 5-year storage period. The results from this study demonstrate that previous grinding and extended storage did not significantly alter white snakeroot toxicity. The results also indicate that tremetone concentration is not the singular indicator of toxicity and that other white snakeroot toxins or toxic tremetone degradation products remain in dried, stored white snakeroot.

Anagyrine desensitization of peripheral nicotinic acetylcholine receptors. A potential biomarker of quinolizidine alkaloid teratogenesis in cattle.

Anagyrine, a teratogenic quinolizidine alkaloid found in Lupinus spp., is proposed to undergo metabolism by pregnant cattle to a piperidine alkaloid which inhibits fetal movement, the putative mechanism behind crooked calf syndrome. The objective of this study was to test the hypothesis that anagyrine but not lupanine or sparteine can directly, without metabolism, desensitize nicotinic acetylcholine receptors (nAChR) in a cell culture model. SH-SY5Y cells expressing autonomic nAChR, and TE-671 cells expressing fetal muscle-type nAChR were exposed to lupine alkaloids or Dimethylphenylpiperazinium (DMPP) in log10 molar increments from 10nM to 100µM and then to a fixed concentration of acetylcholine (ACh) (10µM for SH-SY5Y cells and 1µM for TE-671 cells) and the responses measured with a membrane potential sensing dye to assess nAChR activation and desensitization. The selective ganglionic nAChR agonist DMPP used as a positive control, was a potent activator and desensitizer of nAChR expressed by SH-SY5Y cells. Lupanine was a weak agonist and desensitizer in SH-SY5Y cells and sparteine was without effect. Anagyrine acted as a partial agonist in both cell lines with EC50 values of 4.2 and 231µM in SH-SY5Y and TE-671 cells, respectively. Anagyrine was a desensitizer of nAChR with DC50 values of 6.9 and 139µM in SH-SY5Y and TE-671 cells, respectively. These results confirm the hypothesis that anagyrine is a potent and effective desensitizer of nAChR, and that anagyrine can directly, without metabolism, desensitize nAChR. Moreover, serum anagyrine concentrations may be a potential biomarker for lupine teratogenicity in cattle.

Activation and Desensitization of Peripheral Muscle and Neuronal Nicotinic Acetylcholine Receptors by Selected, Naturally-Occurring Pyridine Alkaloids.

Teratogenic alkaloids can cause developmental defects due to the inhibition of fetal movement that results from desensitization of fetal muscle-type nicotinic acetylcholine receptors (nAChRs). We investigated the ability of two known teratogens, the piperidinyl-pyridine anabasine and its 1,2-dehydropiperidinyl analog anabaseine, to activate and desensitize peripheral nAChRs expressed in TE-671 and SH-SY5Y cells. Activation-concentration response curves for each alkaloid were obtained in the same multi-well plate. To measure rapid desensitization, cells were first exposed to five potentially-desensitizing concentrations of each alkaloid in log10 molar increments from 10 nM to 100 µM and then to a fixed concentration of acetylcholine (ACh), which alone produces near-maximal activation. The fifty percent desensitization concentration (DC50) was calculated from the alkaloid concentration-ACh response curve. Agonist fast desensitization potency was predicted by the agonist potency measured in the initial response. Anabaseine was a more potent desensitizer than anabasine. Relative to anabaseine, nicotine was more potent to autonomic nAChRs, but less potent to the fetal neuromuscular nAChRs. Our experiments have demonstrated that anabaseine is more effective at desensitizing fetal muscle-type nAChRs than anabasine or nicotine and, thus, it is predicted to be more teratogenic.

The Effect of Co-Administration of Death Camas (Zigadenus spp.) and Low Larkspur (Delphinium spp.) in Cattle.

In many rangeland settings, there is more than one potential poisonous plant. Two poisonous plants that are often found growing simultaneously in the same location in North American rangelands are death camas (Zigadenus spp.) and low larkspur (Delphinium spp.). The objective of this study was to determine if co-administration of death camas would exacerbate the toxicity of low larkspur in cattle. Cattle dosed with 2.0 g of death camas/kg BW showed slight frothing and lethargy, whereas cattle dosed with both death camas and low larkspur showed increased clinical signs of poisoning. Although qualitative differences in clinical signs of intoxication in cattle co-treated with death camas and low larkspur were observed, there were not any significant quantitative differences in heart rate or exercise-induced muscle fatigue. Co-treatment with death camas and low larkspur did not affect the serum zygacine kinetics, however, there was a difference in the larkspur alkaloid kinetics in the co-exposure group. Overall, the results from this study suggest that co-exposure to death camas and low larkspur is not significantly more toxic to cattle than exposure to the plants individually. The results from this study increase our knowledge and understanding regarding the acute toxicity of death camas and low larkspur in cattle.

Interactions Between Bacteria and the Gut Mucosa: Do Enteric Neurotransmitters Acting on the Mucosal Epithelium Influence Intestinal Colonization or Infection?

The intestinal epithelium is a critical barrier between the internal and external milieux of the mammalian host. Epithelial interactions between these two host environments have been shown to be modulated by several different, cross-communicating cell types residing in the gut mucosa. These include enteric neurons, whose activity is influenced by bacterial pathogens, and their secreted products. Neurotransmitters appear to influence epithelial associations with bacteria in the intestinal lumen. For example, internalization of Salmonella enterica and Escherichia coli O157:H7 into the Peyer's patch mucosa of the small intestine is altered after the inhibition of neural activity with saxitoxin, a neuronal sodium channel blocker. Catecholamine neurotransmitters, such as dopamine and norepinephrine, also alter bacterial internalization in Peyer's patches. In the large intestine, norepinephrine increases the mucosal adherence of E. coli. These neurotransmitter actions are mediated by well-defined catecholamine receptors situated on the basolateral membranes of epithelial cells rather than through direct interactions with luminal bacteria. Investigations of the involvement of neuroepithelial communication in the regulation of interactions between the intestinal mucosa and luminal bacteria will provide novel insights into the mechanisms underlying bacterial colonization and pathogenesis at mucosal surfaces.

Differences between Angus and Holstein cattle in the Lupinus leucophyllus induced inhibition of fetal activity.

Calves with congenital defects born to cows that have grazed teratogenic Lupinus spp. during pregnancy can suffer from what is termed crooked calf syndrome. Crooked calf syndrome defects include cleft palate, spinal column defects and limb malformations formed by alkaloid-induced inhibition of fetal movement. In this study, we tested the hypothesis that there are differences in fetal activity of fetuses carried by Holstein verses Angus heifers orally dosed with 1.1 g/kg dried ground Lupinus leucophyllus. Fetal activity was monitored via transrectal ultrasonography and maternal serum was analyzed for specific lupine alkaloids. There were more (P < 0.05) movements in fetuses of Holstein heifers than those in Angus heifers at eight and 12 h after oral dosing. In addition to serum alkaloid toxicokinetic differences, the Holstein heifers had significantly lower serum concentrations of anagyrine at 2, 4, and 8 h after oral dosing than Angus heifers. Holstein heifers also had significantly greater serum concentrations of lupanine at 12, 18 and 24 h after dosing than the Angus heifers. These results suggest that there are breed differences in susceptibility to lupine-induced crooked calf syndrome. These differences may also be used to discover genetic markers that identify resistant animals, thus facilitating selective breeding of resistant herds.

The non-competitive blockade of GABAA receptors by an aqueous extract of water hemlock (Cicuta douglasii) tubers.

Water hemlocks (Cicuta spp.) are acutely toxic members of the Umbellierae family; the toxicity is due to the presence of C17-polyacetylenes such as cicutoxin. There is only limited evidence of noncompetitive antagonism by C17-polyacetylenes at GABAA receptors. In this work with WSS-1 cells, we documented the noncompetitive blockade of GABAA receptors by an aqueous extract of water hemlock (Cicuta douglasii) and modulated the actions of the extract with a pretreatment of 10 µM midazolam.