Competitive exclusion of Yersinia enterocolitica biotype 4, serotype O:3 by Yersinia enterocolitica biotype 1A,serotype O:6,30 in tissue culture and in pigs

AIMS: To study the adhesion properties of a biotype 4, serotype O:3 (human pathogenic) strain of Yersinia enterocolitica and to determine if adhesion in vitro and colonisation in vivo can be prevented by competition with a biotype 1A, serotype O:6,30 (non-pathogenic) strain. To study interaction between Y. enterocolitica biotype 4, serotype O:3 and cultured epithelial cells using the synthetic tripeptide arginine-glycine-aspartic acid (RGD).

METHODS: The human intestinal epithelial (HEp-2) cell line was used for in vitro studies. Inocula of Y. enterocolitica biotype 4, serotype O:3 radiolabelled using tritium were incubated with HEp-2 cells and RGD tripeptide, or with Y. enterocolitica biotype 1A, serotype O:6,30 sequentially or concurrently, then washed and lysed, and radioactivity measured to determine the effect of RGD on adhesion, and competitive exclusion of pathogenic by non-pathogenic bacteria. For in vivo studies, two groups of 5-week-old piglets (n=5/group) were sequentially inoculated orally with 5x10⁹ colony forming units (cfu) of either a non-pathogenic biotype 1A, serotype O:6,30 strain of Y. enterocolitica followed by a pathogenic biotype 4, serotype O:3 strain, or vice versa. Pigs were monitored for carriage of strains using bacterial culture and a multiplex polymerase chain reaction (PCR).

RESULTS: The RGD tripeptide significantly inhibited adherence of the pathogenic Y. enterocolitica strain to cultured epithelial cells, suggesting that adhesion involved the RGD tripeptide sequence. The non-pathogenic biotype 1A, serotype O:6,30 strain of Y. enterocolitica prevented adhesion of the pathogenic strain to cells in vitro when allowed to adhere first. Pathogenic Y. enterocolitica was consistently isolated from rectal swabs from 80-100% of pigs on all sampling occasions but not from oral swabs after 14 days in pigs first inoculated with the non- pathogenic strain or at 26 days in pigs first inoculated with the pathogenic strain.

CONCLUSIONS: A non-pathogenic strain of Y. enterocolitica reduced adhesion of a human pathogenic strain in vitro but not in vivo.     

Correlations between ground and excited state spectra of a quantum Dot

The ground and excited state spectra of a semiconductor quantum dot with successive electron occupancy were studied with linear and nonlinear magnetoconductance measurements. A direct correlation was observed between the mth excited state of the N-electron system and the ground state of the (N + m)-electron system for m up to 4. The results are consistent with a single-particle picture in which a fixed spectrum of energy levels is successively filled, except for a notable absence of spin degeneracy. Further departures from the single-particle picture due to electron-electron interaction were also observed. Magnetoconductance fluctuations of ground states show anticrossings where wave function characteristics are exchanged between adjacent levels.     

Transitions between blocked and zonal flows in a rotating annulus with topography

The mid-latitude atmosphere is dominated by westerly, nearly zonal flow. Occasionally, this flow is deflected poleward by blocking anticyclones that persist for 10 days or longer. Experiments in a rotating annulus used radial pumping to generate a zonal jet under the action of the Coriolis force. In the presence of two symmetric ridges at the bottom of the annulus, the resulting flows were nearly zonal at high forcing or blocked at low forcing. Intermittent switching between blocked and zonal patterns occurs because of the jet's interaction with the topography. These results shed further light on previous atmospheric observations and numerical simulations.     

Quantification of insulin-like growth factor binding protein mRNA using real-time PCR in bovine granulosa and theca cells: effect of estradiol, insulin, and gonadotropins

The effects of estradiol, insulin, and gonadotropins on levels of insulin-like growth factor binding protein (IGFBP)-2, -3, -4, and -5 mRNA levels in bovine granulosa and theca cells were evaluated in vitro using serum-free medium containing various hormone treatments arranged in four different experiments. Amounts of IGFBP-2, -3, -4 and -5 mRNA were quantitated using fluorescent quantitative real-time RT-PCR. In small-follicle (1-5 mm) granulosa cells, follicle-stimulating hormone (FSH) in the presence or absence of insulin increased (P<0.05) IGFBP-3 mRNA but did not change IGFBP-2, -4, or -5 mRNA levels; estradiol was without effect on IGFBP-2, -3, -4, or -5 mRNA levels in the absence of insulin but increased (P<0.05) IGFBP-2 mRNA levels in the presence of insulin. Luteinizing hormone (LH) in the absence (but not presence) of insulin increased (P<0.05) small-follicle granulosa cell IGFBP-3 mRNA levels. In large-follicle (>7.9 mm) granulosa cells, insulin alone increased (P<0.05) IGFBP-2 gene expression while LH, FSH, and estradiol were without effect (P>0.10). Estradiol (3 and 300 ng/ml) decreased (P<0.05) IGFBP-5 mRNA levels in large-follicle granulosa cells. In theca cells, insulin decreased (P<0.05) IGFBP-4 expression, but had no effect (P>0.10) on IGFBP-2, -3, or -5 mRNA levels. Estradiol decreased (P<0.05) IGFBP-2, -3, and -4 mRNA levels but had no effect on IGFBP-5 mRNA levels in theca cells. LH had no effect on levels of IGFBP-2, -3, -4, or -5 mRNA in theca cells. These results indicate that expression of IGFBP-2, -3, -4, and -5 mRNA by granulosa and theca cells are differentially regulated by estradiol, insulin and gonadotropins, therefore discretely modulating the amount of bioavailable IGFs to these cells depending upon the specific hormonal stimuli. In particular, these studies are the first in cattle to show that estradiol selectively inhibits IGFBP-2, -3, and -4 gene expression in theca cells, inhibits IGFBP-5 gene expression in large-follicle granulosa cells, and stimulates IGFBP-2 gene expression in small-follicle granulosa cells.     

Proteolysis of insulin-like growth factor binding proteins during preovulatory follicular development in cattle

The present study was conducted to evaluate changes in follicular fluid (FF) insulin-like growth factor binding protein (IGFBP) proteolytic activity and levels of steroids and IGFBP during follicular development in cattle. Estrous cycles of cows were synchronized with two injections of prostaglandin F2alpha (PGF) 11 d apart and follicular growth monitored via daily rectal ultrasonography in order to identify the dominant follicle. All cows were ovariectomized 48 hr after the second injection of PGF. Follicular fluid was collected individually for all follicles > 5 mm and pooled for small (1 to 5 mm) follicles. Follicular fluid estradiol and androstenedione levels were greater (P < 0.05) and progesterone and IGFBP-3 levels not different (P > 0.10) in large dominant than in small (1 to 5 mm) or large (>5 mm) subordinate follicles, whereas IGFBP-2, -4 and -5 levels were less (P < 0.05) in large dominant than in small or large subordinate follicles. To evaluate proteolysis of IGFBPs, FF was incubated with recombinant human (125) I-labeled IGFBP-2, -3, -4, and -5 and proteins separated by 12% SDS-PAGE. Follicular fluid caused little or no proteolysis of (125)I-lableled IGFBP-2 or -3. However, cleavage of (125)I-labeled IGFBP-4 and -5 by FF from large dominant follicles was greater (P < 0.05) than by FF from small or large subordinate follicles indicating that a protease to IGFBP-4 and -5 exists in estrogen dominant follicles. We conclude that lower levels of IGFBP-2 in estrogen dominant follicles of cattle are not due to increased proteolysis, whereas decreases in IGFBP-4 and -5 levels are likely due, in part, to increased protease activity. Changes in IGFBP may alter levels of bioavailable IGFs that stimulate steroidogenesis and mitogenesis in developing bovine follicles.