Kinetics of phase variation between S and type-1 fimbriae of Escherichia coli

Abstract The rate of fimbrial phase variation in Escherichia coli strain 3040 was determined. The strain has type-1 and S fimbriae. The bacterial culture was fractionated into homogeneous subpopulations expressing either one of the fimbrial types only; the subpopulations were inoculated into broth and the fimbriation of individual cells was assayed by immunofluorescence as a function of time. The rate of the shift from S- or type-1-fimbriate cells to non-fimbriate ones was of the order of 10⁻² per cell generation and more rapid than a direct shift from one fimbrial phase to another, although both types of phase variations were observed.     

Fractionation of a bacterial cell population by adsorption to erythrocytes and yeast cells

Abstract Fluorochrome-labeled antibodies specific for either S or type-1 fimbriae of Escherichia coli were used to show that in broth culture the two fimbrial types of strain 3040 mostly occurred on different cells. 12% of the cells were nonfimbriated. A fractionation procedure that involved adsorption of bacterial cells onto erythrocytes and yeast cells was developed to isolate homogeneous subpopulations (S-fimbriated, type-1-fimbriated, and non-fimbriated) of. E. coli. The level of contamination in each isolated subpopulation was 4% at the highest. The method is useful in obtaining homogeneous bacterial populations for adherence studies and for purification of specific fimbrial antigens.     

A TGF-β type I receptor-like molecule with a key functional role in Haemonchus contortus development

Here we investigated the gene of a transforming growth factor (TGF)-β type I receptor-like molecule in Haemonchus contortus, a highly pathogenic and economically important parasitic nematode of small ruminants. Designated Hc-tgfbr1, this gene is transcribed in all developmental stages of H. contortus, and the encoded protein has glycine-serine rich and kinase domains characteristic of a TGF-β family type I receptor. Expression of a GFP reporter driven by the putative Hc-tgfbr1 promoter localised to two intestinal rings, the anterior-most intestinal ring (int ring I) and the posterior-most intestinal ring (int ring IX) in Caenorhabditis elegans in vivo. Heterologous genetic complementation using a plasmid construct containing Hc-tgfbr1 genomic DNA failed to rescue the function of Ce-daf-1 (a known TGF-β type I receptor gene) in a daf-1-deficient mutant strain of C. elegans. In addition, a TGF-β type I receptor inhibitor, galunisertib, and double-stranded RNA interference (RNAi) were employed to assess the function of Hc-tgfbr1 in the transition from exsheathed L3 (xL3) to the L4 of H. contortus in vitro, revealing that both galunisertib and Hc-tgfbr1-specific double-stranded RNA could retard L4 development. Taken together, these results provide evidence that Hc-tgfbr1 is involved in developmental processes in H. contortus in the transition from the free-living to the parasitic stage.     

Dominance relations in captive groups of adult and juvenile arctic blue foxes (Alopex lagopus)

Dominance relationships were studied in captive Arctic blue fox (Alopex lagopus) groups comprising adults (four males, five females) and juveniles (four males, five females). The results showed that Arctic blue foxes easily formed a social organization with an observable hierarchy, in which adults typically dominated over juveniles. Within the same age group, males usually dominated over females. Dominance correlated most significantly with body weight in autumn, but later that correlation decreased. Urine marking activity was very low during autumn and early winter, but increased significantly prior to and during the breeding season when aggressive encounters were also most pronounced. In addition to several adults, the social status of some juveniles was high at breeding time. Altogether 7 out of 11 females (63.6%) whelped, but the survival rate of litters was low and kits of only two adult females survived (18.2%). It can be concluded that hierarchical development and reproduction in Arctic blue fox groups are markedly influenced by dominance relationships.     

Stocking success of newly hatched pike evaluated by radioactive strontium (85Sr) marking

The stocking of newly hatched pike Esox lucius labelled with radioactive strontium (⁸⁵Sr) and later electrofishing of the same area was found to be a workable tool in assessing the success of pike stockings. ⁸⁵Sr‐labelled individuals constituted 96% of the 0+year pike catch from late summer electrofishing in the stocked littoral areas of the heavily regulated Lokka Reservoir. The density of 0+year pike reached 2·1 individuals 100 m⁻² in the stocked areas in comparison to 0·3 individuals 100 m⁻² in the reference areas. In the three other stocked lakes the proportion of labelled pike in the catch ranged from 0 to 42% in late summer. The results suggest that pike stockings are profitable in filling empty or nearly empty 0+year pike habitats of regulated lakes or other waterbodies, where the natural reproduction of pike has declined due to human activities.     

A robust and reproducible animal serum-free culture method for clinical-grade bone marrow-derived mesenchymal stromal cells

Efficient xenofree expansion methods to replace fetal bovine serum (FBS)-based culture methods are strongly encouraged by the regulators and are needed to facilitate the adoption of mesenchymal stromal cell (MSC)-based therapies. In the current study we established a clinically-compliant and reproducible animal serum-free culture protocol for bone marrow-(BM-) MSCs based on an optimized platelet-derived supplement. Our study compared two different platelet-derived supplements, platelet lysate PL1 versus PL2, produced by two different methods and lysed with different amounts of freeze–thaw cycles. Our study also explored the effect of a low oxygen concentration on BM-MSCs. FBS-supplemented BM-MSC culture served as control. Growth kinetics, differentiation and immunomodulatory potential, morphology, karyotype and immunophenotype was analysed. Growth kinetics in long-term culture was also studied. Based on the initial results, we chose to further process develop the PL1-supplemented culture protocol at 20 % oxygen. The results from 11 individual BM-MSC batches expanded in the chosen condition were consistent, yielding 6.60 × 10⁹ ± 4.74 × 10⁹ cells from only 20 ml of bone marrow. The cells suppressed T-cell proliferation, displayed normal karyotype and typical MSC differentiation potential and phenotype. The BM-MSCs were, however, consistently HLA-DR positive when cultured in platelet lysate (7.5–66.1 %). We additionally show that culture media antibiotics and sterile filtration of the platelet lysate can be successfully omitted. We present a robust and reproducible clinically-compliant culture method for BM-MSCs based on platelet lysate, which enables high quantities of HLA-DR positive MSCs at a low passage number (p2) and suitable for clinical use.     

Associative Nitrogen Fixation by Klebsiella spp.: Adhesion Sites and Inoculation Effects on Grass Roots

Adhesion sites on grass roots for Klebsiella strains carrying type 3 or type 1 fimbriae or both were determined. Adhesion of the strains to the roots of Poa pratensis and Festuca rubra was highly localized; the bacteria adhered strongly to root hairs and with a markedly lower efficiency to the surface of the zone of elongation and to the root cap mucilage. No adhesion to the epidermal cells between root hairs was observed. The adhesion sites were identical for the type 3- and 1-fimbriated bacteria and for P. pratensis, F. rubra, and Trifolium pratense. Inoculation of P. pratensis seedlings with Klebsiella pneumoniae strain As resulted in morphological changes in plant roots. The roots of infected plants were heavily covered with root hairs, which often were deformed and branched.     

Binding of purified Escherichia coli O75X adhesin to frozen sections of human kidney

Abstract Binding characteristics of the purified Escherichia coli O75X adhesin in frozen sections of human kidney were determined, using antibodies raised against the purified antigen and rhodamine-conjugated second antibodies. To identify the adhesin-binding nephron domains, the same tissue sections were double stained with fluorescein isothiocynate-conjugated nephron site-specific lectins. The results revealed that, at the tubular pole, the O75X adhesin bound selectively to the basement membrane of proximal and distal tubules and, with a slightly lower efficiency, of collecting ducts. In the glomerulus, the O75X adhesin bound only to the parietal epithelial cells (Bowman's capsule). The purified O75X adhesin bound also to exfoliated epithelial cells in human urine. Our results suggest that the O75X adhesin may contribute to the uropathogenicity of E. coli by binding the bacteria to tissue structures in the human urinary tract.     

Characterization of type 1 pili of Salmonella typhimurium LT2.

Type 1 pili from Salmonella typhimurium LT2 were purified and characterized. The pilus filaments were 6 nm in diameter and over 1 microns long. Estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of the pilin was 21,000. The isoelectric point of the filament was 4.1. Hydrophobic amino acids comprised 40.3% of the total amino acids of the pilin, which contained more proline, serine, and lysine than reported for the type 1 pilin of Escherichia coli. Purified pili agglutinated both horse and chicken erythrocytes and yeast cells but not bovine, sheep, or human erythrocytes. Horse erythrocyte agglutination was inhibited at lower concentrations by alpha-methyl-D-mannoside than by yeast mannane and D-fructose. Agglutination was not affected by D-galactose or sucrose. Results of the present study confirm the role of type 1 pili as Salmonella hemagglutinins and show chemical differences between the type 1 pili of S. typhimurium and E. coli.