Lactate dehydrogenase release assay from Vero cells to distinguish verotoxin producing Escherichia coli from non-verotoxin producing strains.

The Vero cell assay presently used for virulence testing of verotoxigenic Escherichia coli (VTEC) requires at least 48-96 h where cytotoxicity effects are examined under a microscope. Here, a complimentary rapid assay was developed that measures endogenous lactate dehydrogenase (LDH) release from Vero or HEp-2 cells as an indicator of cytotoxicity. Toxin preparations from 24 VTEC strains induced 36-89% LDH from Vero cells and 15-62% LDH from HEp-2 cells in 12-16 h. A verotoxin-positive but enterohemolysin negative strain also showed a similar cytotoxicity effect. In contrast, three VT-negative strains caused only 13-16% LDH from Vero cells and 1-7% LDH from HEp-2 cells. Five presumptive E. coli isolates from naturally contaminated food and clinical sources did not induce significant LDH release from either cell lines. PCR analysis confirmed the presence of vt1 or vt2 genes in E. coli showing positive LDH values. Similarly, RiboPrinter analysis confirmed and identified the test strains as E. coli except for two meat isolates, which were identified as Hafnia alvei. Cytopathic effects of toxin preparations from VTEC revealed severe lysis, vacuole formation and death in Vero cells and multiple vacuoles and cell elongation in HEp-2 cells. The colorimetric cytotoxicity assay described here can provide quantitative data for determining the virulence potential of verotoxigenic E. coli in 12-16 h.

Behavioral and physiological control of yolk synthesis and deposition in the female red-sided garter snake (Thamnophis sirtalis parietalis).

Ovarian recrudescence in female garter snakes, Thamnophis sirtalis parietalis, follows spring emergence from hibernation and mating. In the laboratory, courtship and mating stimuli significantly increased the proportion of female garter snakes becoming pregnant, although some noncourted nonmated controls also became pregnant. Females given artificial mating stimuli under anesthetic, without courtship stimuli, were no more likely than either noncourted, nonmated or anesthetized controls to become pregnant. Hormonal changes and yolk synthesis rapidly followed mating in both laboratory and field females; serum estradiol increased more than 10-fold in 2 days and serum calcium, a measure of yolk precursor lipoprotein (vitellogenin) concentration, increased more than two times in 10 days. Administration of exogenous estradiol stimulated yolk synthesis, but did not result in yolk deposition into ovarian follicles. However, administration of ovine follicle-stimulating hormone induced both hepatic yolk synthesis and yolk deposition. Our results are consistent with the hypotheses that courtship and copulation are facilitatory to ovarian recrudescence but neither alone nor in combination is necessary nor sufficient, and in this species yolk synthesis and yolk deposition are separately regulated.

Hormonal independence of courtship behavior in the male garter snake.

Garter snakes exhibit a dissociated reproductive tactic in which gonadal activity is minimal at the time of mating, increasing only after the breeding season has ended. Experiments are presented demonstrating that neither short-term nor longterm castration affects courtship behavior in adult male red-sided garter snakes (Thamnophis sirtalis parietalis). So long as males have passed through a low-temperature dormancy period (hibernation), castration either shortly after emergence in the spring, or before entering winter dormancy in the fall, does not prevent the display of intense courtship behavior on emergence. Similarly, males castrated during mating activity the previous spring prior to the annual testicular growth phase actively courted females on emergence from hibernation. Males adrenalectomized and castrated during low-temperature dormancy also courted females on emergence. Hypophysectomy during or before low-temperature dormancy did not prevent males from displaying high-intensity courtship behavior on emergence from hibernation. Treatment with sex steroid hormones, as well as hypothalamic and pituitary hormones, and a variety of neural and metabolic affectors also fails to elicit courtship behavior in noncourting males during the summer. It was concluded that causal mechanisms controlling courtship behavior in the red-sided garter snake are fundamentally different, at least at the physiological level, from those mechanisms described for many laboratory and domesticated species.

Metabolic changes in male snakes, Thamnophis melanogaster, during a breeding period.

1. Daily basking periods were lengthened substantially during a breeding period in a natural population of the snake, Thamnophis melanogaster. 2. In the laboratory, oxygen consumption of male T. melanogaster significantly increased during a breeding period, compared to pre- and post-breeding levels in the same males. 3. In both laboratory and field males, serum lipid increased initially and then decreased during the breeding period. 4. Serum protein of laboratory males decreased significantly following the breeding period. 5. Serum glucose did not change significantly in laboratory males, but increased significantly during the breeding period in field males. 6. Changes in oxygen consumption and nutrient distribution may reflect changing energy demands attendant with gonadal recrudescence in this snake.

Female control of male reproductive function in a Mexican Snake.

Male Thamnophis melanogaster court immediately when exposed to estrogen-treated, attractive females and continue courting for 6 to 8 days. Males exposed to estrogen-treated females will court both intact and ovariectomized females. These males undergo a period of testicular recrudescence, whereas males exposed only to ovariectomized females do no. Sexual attractivity can be induced in female T. melanogaster without estrogen treatment by heavy feeding, which results in significant increases in liver size and activity.

Female sex pheromone in the skin and circulation of a garter snake.

Serums and extracts of tissues from the female garter snake (Thamnophis sirtalis parietalis) each act as a pheromone and elicit male courtship behavior when applied to the back of another male. Since pheromonal activity is present in yolk and liver tissue of untreated females and can be induced with estrogen treatment in the serums and livers of males, the pheromone may be associated with the circulating yolk lipoprotein, vitellogenin.