Kip E. Panter

Multiple congenital contractures (mcc) and cleft palate induced in goats by ingestion of piperidine alkaloid-containing plants: Reduction in fetal movement as the probable cause

Fetal movement, observed by ultrasound imaging, was significantly reduced (P less than or equal to 0.001) in pregnant goats gavaged with Conium seed and Nicotiana glauca and temporarily reduced with fresh Conium plant. Conium seed and Nicotiana glauca induced cleft palate and multiple congenital contractures in 100% of the kids born to pregnant goats gavaged with these plants. Multiple congenital contractures included torticollis, scoliosis, lordosis, arthrogryposis, rib cage anomalies, over extension, and flexure and rigidity of the joints. However, in goats gavaged with fresh Conium plant, fetal movement was inhibited for only about 5 hours after each individual dosage and gradually returned to control levels 12 hours after dosing. Fetal malformations in this group were limited from modest to moderate contractures of the front limbs, which resolved by 8-10 weeks post partum. No cleft palates were induced. Fetal movement was not inhibited in goats fed Lupinus caudatus and no cleft palates or multiple congenital contractures were induced in their offspring. The duration of the reduction in fetal movement appears to be an important factor in the severity and permanence of the deformities, particularly with cleft palate, spinal column defects, and severe joint deviation and fixation.

Comparative Toxicosis of Sodium Selenite and Selenomethionine in Lambs

Excess consumption of selenium (Se) accumulator plants can result in selenium intoxication. The objective of the study reported here was to compare the acute toxicosis caused by organic selenium (selenomethionine) found in plants with that caused by the supplemental, inorganic form of selenium (sodium selenite). Lambs were orally administered a single dose of selenium as either sodium selenite or selenomethionine and were monitored for 7 days, after which they were euthanized and necropsied. Twelve randomly assigned treatment groups consisted of animals given 0, 1, 2, 3, or 4 mg of Se/kg of body weight as sodium selenite, or 0, 1, 2, 3, 4, 6, or 8 mg of Se/kg as selenomethionine. Sodium selenite at dosages of 2, 3, and 4 mg/kg, as well as selenomethionine at dosages of 4, 6, and 8 mg/kg resulted in tachypnea and/or respiratory distress following minimal exercise. Severity and time to recovery varied, and were dose dependent. Major histopathologic findings in animals of the high-dose groups included multifocal myocardial necrosis and pulmonary alveolar vasculitis with pulmonary edema and hemorrhage. Analysis of liver, kidney cortex, heart, blood, and serum revealed linear, dose-dependent increases in selenium concentration. However, tissue selenium concentration in selenomethionine-treated lambs were significantly greater than that in lambs treated with equivalent doses of sodium selenite. To estimate the oxidative effects of these selenium compounds in vivo, liver vitamin E concentration also was measured. Sodium selenite, but not selenomethionine administration resulted in decreased liver vitamin E concentration. Results of this study indicate that the chemical form of the ingested Se must be known to adequately interpret tissue, blood, and serum Se concentrations.

Garlic in the ovine fetal environment

We investigated whether the odor of garlic can cross the placental barrier from the mother to the fetal environment. Samples of amniotic fluid, allantoic fluid, fetal blood, and maternal blood were collected 0, 50, 100, and 150 min after a pregnant ewe (approximately day 110 of gestation) was gavaged with 6 ml of Egyptian garlic oil. A panel of judges detected (p < 0.05) garlic odor in samples of allantoic fluid, fetal blood, and maternal blood collected 50, 100, and 150 min after the ewe was given garlic and in samples of amniotic fluid collected 100 min after treatment.

The fetal cleft palate: I. Characterization of a congenital model.

Any animal model of a human congenital anomaly established by iatrogenic methods involving intrauterine fetal manipulation has limited clinical applicability. A congenital model that more closely simulates the etiopathogenesis of a human anomaly may provide data that can more readily be extrapolated to that anomaly and, therefore, be used in diagnostic and management strategies. The present work provides a description and characterization of a congenital model of cleft palate in the goat. Palatal shelf closure normally occurs at approximately day 38 of gestation in the caprine species. Sixteen pregnant goats were gavaged twice daily during gestational days 32 through 41 [term, 145 days] with a plant slurry of Nicotiana glauca containing the piperidine alkaloid teratogen anabasine. Gross analysis and measurement of fetal clefts were performed at 60, 70, and 85 days gestation (four fetuses were studied at each time point). Seventeen clefted kids were sacrificed at specific intervals after birth (2 weeks, and 1, 3, and 6 months); after skull debridement and preparation, they were compared with 12 unclefted control kids. Complete clefting of the secondary palate occurred in 97 percent of the fetuses. In all cases, the cleft extended from the posterior aspect of the alveolar ridge to the uvula; the majority of these clefts were bilateral, with complete detachment of the vomer. Morphologically, these clefts were similar to human clefts. Eighteen percent of clefted newborn kids demonstrated gross maxillary hypoplasia and midfacial retrusion at birth with a relative Class III malocclusion. Direct measurement of the congenital caprine skulls confirmed these findings. The incidence of midfacial growth abnormalities in these clefted animals raises questions regarding the etiopathogenesis of facial dysmorphology that is unrelated to scarring of the maxilla. This congenital cleft palate model is currently being used to explore these questions and others related to craniofacial growth and palatal function after in utero repair.

Congenital skeletal malformations and cleft palate induced in goats by ingestion of Lupinus, Conium and Nicotiana species

Three piperidine alkaloid containing plants, Conium maculatum (poison-hemlock), Nicotiana glauca (tree tobacco) and Lupinus formosus (lunara lupine), induced multiple congenital contractures (MCC) and palatoschisis in goat kids when their dams were gavaged with the plant during gestation days 30-60. The skeletal abnormalities included fixed extension or flexure of the carpal, tarsal, and fetlock joints, scoliosis, lordosis, torticollis and rib cage abnormalities. Clinical signs of toxicity included those reported in sheep, cattle and pigs--ataxia, incoordination, muscular weakness, prostration and death. One quinolizidine alkaloid containing plant, Lupinus caudatus (tailcup lupine), on the other hand, which is also known to cause MCC in cows, caused only slight signs of toxicity in pregnant goats and no teratogenic effects in their offspring.

Dose response of sheep poisoned with locoweed (Oxytropis sericea)

Locoweed poisoning occurs when livestock consume swainsonine-containing Astragalus and Oxytropis species over several weeks. Although the clinical and histologic changes of poisoning have been described, the dose or duration of swainsonine ingestion that results in significant or irreversible damage is not known. The purpose of this research was to document the swainsonine doses that produce clinical intoxication and histologic lesions. Twenty-one mixed-breed wethers were dosed by gavage with ground Oxytropis sericea to obtain swainsonine doses of 0.0, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.0 mg/kg/day for 30 days. Sheep receiving ≥0.2 mg/kg gained less weight than controls. After 16 days, animals receiving ≥0.4 mg/kg were depressed, reluctant to move, and did not eat their feed rations. All treatment groups had serum biochemical changes, including depressed α-mannosidase, increased aspartate aminotransferase and alkaline phosphatase, as well as sporadic changes in lactate dehydrogenase, sodium, chloride, magnesium, albumin, and osmolarity. Typical locoweed-induced cellular vacuolation was seen in the following tissues and swainsonine doses: exocrine pancreas at ≥0.05 mg/kg; proximal convoluted renal and thyroid follicular epithelium at ≥0.1 mg/kg; Purkinjes cells, Kupffers cells, splenic and lymph node macrophages, and transitional epithelium of the urinary bladder at ≥0.2 mg/kg; neurons of the basal ganglia, mesencephalon, and metencephalon at ≥0.4 mg/kg; and cerebellar neurons and glia at ≥0.8 mg/kg. Histologic lesions were generally found when tissue swainsonine concentrations were ≅150 ng/g. Both the clinical and histologic lesions, especially cerebellar lesions are suggestive of neurologic dysfunction even at low daily swainsonine doses of 0.2 mg/kg, suggesting that prolonged locoweed exposure, even at low doses, results in significant production losses as well as histologic and functional damage.

Comparison of cleft palate induction by Nicotiana glauca in goats and sheep.

The induction of cleft palate by Nicotiana glauca (wild tree tobacco) during the first trimester of pregnancy was compared between Spanish-type goats and crossbred western-type sheep. Cleft palate was induced in 100% of the embryonic/fetal goats when their pregnant mothers were gavaged with N. glauca plant material or with anabasine-rich extracts from the latter, during gestation days 32-41. Seventy-five percent of newborn goats had cleft palate after maternal dosing with N. glauca during gestation days 35-41, while no cleft palates were induced when dosing periods included days 36-40, 37-39, or day 38 only. The induced cleft palates were bilateral, involving the entire secondary palates with complete detachment of the vomer. Eleven percent of the newborn goats from does gavaged during gestation days 32-41 had extracranial abnormalities, most often contractures of the metacarpal joints. Most of these contractures resolved spontaneously by 4-6 weeks postpartum. One newborn kid also had an asymmetric skull due to apparent fetal positioning. No cleft palates were induced in lambs whose mothers were gavaged with N. glauca plant or anabasine-rich extracts during gestation days 34-41, 35-40, 35-41, 36-41, 35-51, or 37-50. Only one of five lambs born to three ewes gavaged with N. glauca plant material during gestation days 34-55 had a cleft palate, but all five of these lambs had moderate to severe contractures in the metacarpal joints. The slight to moderate contracture defects resolved spontaneously by 4-6 weeks postpartum, but the severe contractures resolved only partially. Embryonic/fetal death and resorption (determined by ultrasound) occurred in 25% of pregnant goats fed N. glauca compared to only 4% of pregnant sheep. Nicotiana glauca plant material contained the teratogenic alkaloid anabasine at 0.175% to 0.23%, dry weight, demonstrating that Spanish-type goats are susceptible to cleft palate induction by the natural toxin anabasine, while crossbred western-type sheep are resistant. However, clinical signs of toxicity were equally severe in goats and sheep, even though maternal alkaloid tolerance was generally lower in sheep. We postulate that an alkaloid-induced reduction in fetal movement during the period of normal palate closure is the cause of the cleft palate and multiple flexion contractures. Teratology 61:203-210, 2000. Published 2000 Wiley-Liss, Inc.

Management of three toxic Delphinium species based on alkaloid concentrations

A systematic approach to the taxonomic classification of the tall larkspur complex (Delphinium spp.) has been developed and implemented using molecular genetics, plant morphology, and alkaloid profiles, as shown in other papers in this series. This approach supports the classification of three distinct species ( D. glaucum, D. barbeyi and D. occidentale), as the species differ in genetics and toxicity. Toxic alkaloid concentrations over the growing season were integrated with data on diet selection to make management recommendations on a species-specific basis to reduce the risk of poisoning cattle. Alkaloid concentrations in tall larkspurs in excess of 3 mg/g impart moderate or high risk to grazing cattle if sufficient quantities are consumed. D. glaucum is most toxic, with toxic alkaloid concentrations that exceed 3 mg/g throughout the grazing season until late maturity. Cattle should be denied access to dense patches of this species throughout the grazing season until after seed shatter. Concentration of toxic alkaloids in D. barbeyi is highest in vegetative plants, but D. barbeyi is unpalatable to cattle until flowering racemes begin to elongate. We recommend grazing D. barbeyi ranges early in the season when it is not palatable, then removing cattle from early flowering stage through mid-pod stage when cattle are most likely to be poisoned. Cattle can again safely graze D. barbeyi late in the season when the toxic alkaloid concentration typically declines below 3 mg/g. Some populations of D. occidentale and the D. barbeyi× D. occidentale hybrids do not contain toxic alkaloids, and pose little risk of poisoning throughout the year. Toxicity of northern populations of D. occidentale varies from year-to-year for unknown reasons. Cattle

Stereoselective Potencies and Relative Toxicities of Coniine Enantiomers

Coniine, one of the major toxic alkaloids present in poison hemlock ( Conium maculatum), occurs in two optically active forms. A comparison of the relative potencies of (+)- and (-)-coniine enantiomers has not been previously reported. In this study, we separated the enantiomers of coniine and determined the biological activity of each enantiomer in vitro and in vivo. The relative potencies of these enantiomers on TE-671 cells expressing human fetal nicotinic neuromuscular receptors had the rank order of (-)-coniine > (+/-)-coniine > (+)-coniine. A mouse bioassay was used to determine the relative lethalities of (-)-, (+/-)-, and (+)-coniine in vivo. The LD 50 values of the coniine enantiomers were 7.0, 7.7, and 12.1 mg/kg for the (-)-, (+/-)-, and (+)- forms of coniine, respectively. The results from this study demonstrate that there is a stereoselective difference in the in vitro potencies of the enantiomers of coniine that directly correlates with the relative toxicities of the enantiomers in vivo.

Cyclopamine: From Cyclops Lambs to Cancer Treatment

In the late 1960s, the steroidal alkaloid cyclopamine was isolated from the plant Veratrum californicum and identified as the teratogen responsible for craniofacial birth defects including cyclops in the offspring of sheep grazing on mountain ranges in the western United States. Cyclopamine was found to inhibit the hedgehog (Hh) signaling pathway, which plays a critical role in embryonic development. More recently, aberrant Hh signaling has been implicated in several types of cancer. Thus, inhibitors of the Hh signaling pathway, including cyclopamine derivatives, have been targeted as potential treatments for certain cancers and other diseases associated with the Hh signaling pathway. A brief history of cyclopamine and cyclopamine derivatives investigated for the treatment of cancer is presented.