Azotobacter strains require molybdenum while utilizing N2 as a source of nitrogen for growth (Bortels, 1930, Homer, et al., 1942, Mulder, 1948, Jensen, 1947, Keeler, 1955). This requirement has prompted us to investigate the distribution of molybdenum in fractions obtained by differential centrifugation of cell-free extracts of Azotobacter vinelandii. Assuming molybdenum to be a constituent of the nitrogen-fixing system, it appeared that fractionation might yield a particulate fraction upon which one could center attention in cell-free nitrogen fixation studies. This paper reports the isolation of a molybdenum-rich particulate fraction which contains most of the molybdenum taken up by the growing cells and which has a molybdenum to protein ratio higher than any other fraction.

Quinolizidine and piperidine alkaloid teratogens from Lupinus, Conium, and Nicotiana genera have been identified as causes of birth defects in livestock induced by poisonous plants. Many defects now known to be related to poisonous plant ingestion were once thought to have a genetic origin. This supposition delayed diagnosis, reporting, and understanding of such birth defects, because breeders and producers feared the news would make it difficult to sell breeding stock. Defects caused by quinolizidine and piperidine teratogens include cleft palate and contracture-type skeletal defects such as arthrogryposis, scoliosis, torticollis, and kyphosis. Teratogens have been identified, differences in susceptibility to teratogenic compounds among livestock species have been elucidated, periods of gestation when specific types of birth defects occur have been determined, and information about mechanism of action has been developed.

Hamster teratogenicity induced upon oral administration of steroidal alkaloids (solanidanes, spirosolanes and jervanes) appears to relate very closely to the presence or absence of C-5, C-6 unsaturation in the alkaloid, which may be more important than molecular configuration at C-22 and placement of the nitrogen atom with regard to the plane of the steroid.

Many poisonous plants grazed by livestock on ranges and pastures in the western USA are fetotoxic causing fetal malformations, embryonic or fetal death, abortion, or early parturition. Decreased incidence of plant-induced livestock malformations may be accomplished through grazing management strategies. To develop these strategies one must understand some basic principles of toxicology and teratology such as susceptible livestock species, type of compound and concentration in the plant and its disposition in the animal, dose response, and the susceptible gestational period. Much of this information is known for certain plants; however, additional information will enhance our ability to control livestock losses from these plants. Certain criteria may be established to maximize grazing management methods to minimize teratogenic effects of poisonous plants. When the suspect plant grows in a restricted habitat, poses a hazard only at certain growth stages or when the susceptible period of pregnancy is relatively short, minor adjustments in management methods can be considerably successful in reducing incidence of malformations and subsequent financial loss.

Verbesina encelioides administered to sheep by gavage induced clinical signs of toxicity and pathologic lesions identical to those induced by Galega officinalis. Sheep had compromised respiratory function with shallow, rapid respiration and frothy exudate from the nares. Affected animals necropsied at time of death presented with hydrothorax with as much as 2 to 3 L of straw-colored thoracic fluid with fibrin tags and congestion and edema of the lungs. The trachea and lung airways contained frothy material with fibrin strands. In some cases, subendocardial hemorrhage of the left ventricle was present. Galegine, a guanidine compound believed to be responsible for these effects, was found at an average concentration of about 0.46% in Galega and at 0.08% in the Verbesina collection that induced toxicosis. While G. officinalis is a known poisonous plant, its very limited distribution in the U.S. causes it to be of minor importance. V. encelioides, however, is widely distributed in the U.S. and presents a potential hazard for grazing livestock. Verbesina may have been responsible for past livestock deaths in the U.S., and thus should be classified as a poisonous plant.

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.

Six heifer calves were administered the dried and ground whole plant of Thermopsis montana by gavage once daily for 2 to 4 days at an initial dosage rate of 1 g/kg of body weight. Two of the 6 heifers died after the second dose, and the remaining 4 calves were humanely killed and their tissues were examined. Serum creatine kinase and aspartate transaminase activities were significantly (P less than 0.05) increased after initiation of plant administration. All calves had skeletal muscular degeneration or evidence of regeneration and repair of damaged myofibers in all skeletal muscle groups examined. Myoglobinuria and cardiac muscle degeneration were not detected in any of the calves.

Jervine, a steroidal alkaloid found as a minor constituent in the teratogenic range plant Veratrum californicum, has produced similar terata in sheep, rabbit, hamster, and chick, although the sensitivity to the alkaloid varies in the different species. Sprague Dawley rats and Swiss Webster mice are relatively insensitive. The aim of this study was to determine the teratogenic potential of jervine in three strains of mice and to ascertain if the response is strain dependent. One strain, Swiss N:GP(S), was retested since a Swiss Webster strain had been found previously to be jervine-resistant. In addition, we tested C57BL/6J and A/J, which are known to differ in their response to the teratogenic action of steroids and vitamin A. Mice were treated by gavage with single doses of jervine (70, 150, or 300 mg/kg body weight) on either day 8, 9, or 10 of gestation. Jervine was teratogenic to C57BL/6J and A/J mice but not to N:GP(S). The induced terata included cleft lip with or without cleft palate, isolated cleft palate, mandibular micrognathia or agnathia, and limb malformations. Fetal teratogenicity and maternal and fetal toxicity were highly correlated. The prevalence of each defect and fetal death was a function of strain, dose, and time of treatment. Maternal death was higher in C57BL/6J than in A/J mice. Although some of the terata were similar, the response pattern between strains was different from corticosteroids and vitamin A for both sensitive period and the strain dose response. An effect on differentiation of chondrocyte precursors may account for many of the defects, but an earlier lethal effect on differentiation of neural crest cells or precordal mesenchyme may also occur.

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.

Ephe&a vMdi& and Ephe&a nevadknh were gw8ged at high doea to pregnant livestock to determine whether there existed any maternal or embryo/letal toxicity. E. viricls was tested in both sheep and cows and waa toxic, and generally induced rumhl impaction, diarrhea, vomition or anorexia. E. m!va&nsis WY tested only in sheep, but wry devoid of toxicity. Neither plant induced apparent adverse effecta in unboro offiprhg of animal8 paged. They were normal at birth.

A congenital deformity condition called crooked calf disease, of widespread occurrence in western North America, is known to be induced by maternal ingestion during gestation of certain members of the Lupinus genus containing the quinolizidine alkaloid teratogen anagyrine. Because some piperidine alkaloids from other sources induce a similar condition, we have investigated the alkaloid composition and teratogenicity of Lupinus formosus, reported by others to be low in quinolizidines but rich in the type of piperidine alkaloids that we have speculated would be teratogenic. GC/MS analysis of L. formosus showed seven major and nine minor components in the total alkaloid fraction. All seven major and five of the nine minor components, representing all but 3% of the fraction, were identified by mass spectrometric fragmentation patterns and GC retention times. They included several potentially teratogenic piperidine alkaloids (including a very large amount of ammodendrine), as well as several nonteratogenic quinolizidine alkaloids plus a trace (at nonteratogenic levels) of the known quinolizidine teratogen anagyrine. The plant induced severe crooked calf disease with limb, spinal, and palate involvement in experimental calves. The deformities are believed to have been induced by ammodendrine.

Poison hemlock (Conium maculatum) was toxic to pregnant ewes and their fetuses when fed during gestation days 30 through 60. Maternal effects included trembling, muscular weakness in the neck initially, then progressing to the limbs, ataxia, frequent urination and defecation, and death. Convulsive seizures were not observed. Fetotoxic effects included excessive flexure of the carpal joints with lateral deviation in the front limbs and kinked tails. At term, 7 of 11 lambs had varying degrees of the limb abnormalities, but all lambs appeared clinically normal at 8 weeks after parturition.

The hemlocks, Conium maculatum (poison-hemlock) and Cicuta spp. (waterhemlock), are poisonous plants that cause sizeable losss to the livestock industry. Clinical signs of poisonhemlock toxicosis are similar in all species of livestock and include muscular weakness, incordination, trembling, initial central nervous system stimulation, depression and death from respiratory paralysis. Poison-hemlock also causes skeletal defects in the offspring of cattle, pigs and sheep and cleft palate in pigs when ingested during specific periods of gestation. The primary toxicants in poison-hemlock are coniine and gamma-coniceine. Coniine predominates in mature plants and seed, whereas gamma-coniceine predominates in early growth of the plant. Waterhemlock is the most violently toxic poisonous plant known. The toxicant is cicutoxin, which acts on the central nervous system, causing violent convulsions and death. Clinical signs of poisoning appear within 15 min after ingestion of a lethal dose and include excessive salivation, nervousness, tremors, muscular weakness and convulsive seizures interspersed by intermittent periods of relaxation and a final paralytic seizure resulting in anoxia and death. Elevated activities of lactic dehydrogenase, aspartate aminotransferase and creatine kinase in blood are observed, indicative of muscular damage. Toxicoses from poisonhemlock and waterhemlock generally occur in early spring when both plants emerge before other, more palatable plants begin to grow. All parts of the poison-hemlock plant are toxic. The root or tubers of waterhemlock are toxic; however, experimental evidence concerning the toxicity of other plant parts is inconclusive.