Evaluation of chromogenic technologies for use in Australian potable water.

AIMS: To compare the use of MI agar, Membrane Lactose Glucoronide Agar (MLGA), CM1046 agar and Colilert-18 (Defined Substrate Technology, IDEXX Laboratories Pty. Ltd., Sydney) on Australian potable water. METHODS AND RESULTS: Both potable (n = 369) and nonpotable waters (n = 35) were analysed by membrane filtration using chromogenic agars as well as Colilert-18 over a period of 12 months. Recoveries of stressed organisms on these chromogenic media were also investigated. Agar-based chromogenic technologies compared favourably to Colilert-18 for chlorinated waters, but there are possible limitations when using these agars for chloraminated waters. Additionally, the breakthrough of problematic organisms, especially oxidase positive organisms, may lead to misrepresentation or over-estimation of E. coli and total coliforms, particularly on MLGA and CM1046. The recovery of stressed organisms was favoured in the Colilert-18 system when compared to chromogenic agars. CONCLUSIONS: MI agar performed better than the other chromogenic agars with respect to recovery and colour identification and discrimination of organisms, and compared favourably with Colilert-18. The use of chromogenic agars in chloraminated waters should be done cautiously. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides comparison data for laboratories looking to adopt chromogenic technologies, and is especially important for Australian laboratories wanting to uptake the use of MI agar (as used in USEPA method 1604) for routine use and for gaining accreditation. Additionally, to the best of our knowledge, this is the first reported evaluation of these agars in chloraminated waters and is especially timely as the use of this disinfection agent is increasing.

Combined anti-vascular cell adhesion molecule-1 and anti-leukocyte function-associated molecule-1 monoclonal antibody therapy does not prolong allograft survival in an ovine model of renal transplantation.

This study investigates the therapeutic efficacy of an anti-vascular cell adhesion molecule (VCAM)-1 monoclonal antibody (mAb), alone or in combination with an anti-leukocyte function-associated-1 mAb, in prolonging allograft survival in an ovine model of renal transplantation. The kinetics of VCAM-1 induction and expression during renal allograft rejection have also been studied. Sheep receiving anti-ovine VCAM-1 antibody demonstrated graft failure at a mean of 8.4 (+/- SD; 0.7) days after transplantation compared with 9.3 (+/- 0.5) days after transplantation for the group given control antibody and 7.7 (+/- 0.3) days after transplantation in the animals given the combined anti-VCAM-1 and anti-leukocyte function-associated-1 mAb therapy. VCAM-1 expression was detected in the allografts at day 1 after transplantation, with peak expression detected by day 5. Tubular expression of VCAM-1 was minimal, with sparse focal staining at the basolateral surfaces. The degree of mononuclear cell infiltrate in the allografts paralleled the progressive increase in VCAM-1 expression after transplantation, and there was no difference in the level of mononuclear cell infiltrate compared with controls.

Profiling bacterial survival through a water treatment process and subsequent distribution system.

AIMS: To profile fractions of active bacteria and of bacteria culturable with routine heterotrophic plate count (HPC) methods through a typical water treatment process and subsequent distribution system. In doing so, investigate how water treatment affects both bacterial abundance and diversity, and reveal the identities of active bacteria not detected by traditional HPC culture. METHODS AND RESULTS: Profiling active fractions was performed by flow cytometric cell sorting of either membrane-intact (BacLight kit) or enzymatically active (carboxyfluorescein diacetate, CFDA) bacteria, followed by eubacterial 16S rDNA-directed PCR and denaturing gradient gel electrophoresis (DGGE). Water treatment significantly reduced active bacterial numbers detected by the BacLight kit and CFDA assay by 2.89 and 2.81 log respectively. Bacterial diversity was also reduced from > 20 DGGE bands in the active fractions of reservoir water to only two bands in the active fractions of finished water. These two bands represented Stenotrophomonas maltophila, initially culturable by HPC, and a Burkholderia-related species. Both species maintained measurable traits of physiological activity in distribution system bulk water but were undetected by HPC. CONCLUSIONS: Flow cytometric cell sorting with PCR-DGGE, to assess water treatment efficacy, identified active bacteria from a variety of major phylogenetic groups undetected by routine HPC. Following treatment S. maltophila and a Burkholderia-related species retained activity and entered distribution undetected by HPC. SIGNIFICANCE AND IMPACT OF THE STUDY: Methods used here demonstrate how water treatment operators can better monitor water treatment plant efficacy and assess distribution system instability by the detection and identification of active bacteria recalcitrant to routine HPC culture.

Characterization of ovine umbilical vein endothelial cells and their expression of cell adhesion molecules: comparative study with human endothelial cells.

This paper reports the isolation and characterization of sheep umbilical vein endothelial cells (ShUVEC) and examines the kinetics of the expression of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). In culture, the cells demonstrate the classical endothelial cell phenotype, including the expression of von Willebrand Factor (vWF), the metabolism of acetylated-low density lipoprotein (Ac-LDL) and the typical cobblestone monolayer appearance. Northern analysis of the expression of ICAM-1 and VCAM-1 mRNA by ShUVEC stimulated with either LPS or recombinant ovine (ro)-TNF-alpha or IL-1 beta demonstrated peak RNA expression for both molecules at between 2 and 6 h after stimulation. Maximum VCAM-1 protein expression occurred between 6 and 12 h after stimulation with ro-TNF-alpha, followed by a slight decrease to a plateau extending beyond 48 h. The levels and kinetics of expression of additional cell surface proteins on ShUVEC, including P-selectin (CD62P), beta 1 integrin (CD29) and ovine MHC classes I and II, were found to be almost identical to that observed on human endothelial cells (HUVEC) cultured under similar conditions. Based on the above results, we conclude that the ShUVEC represent the ovine equivalent of HUVEC, both phenotypically and functionally.

Anti-ovine VCAM-1 monoclonal antibodies inhibit adhesion and proliferation between sheep endothelial and mononuclear cells in vitro.

This paper reports the production and characterization of three monoclonal antibodies (mAb) recognizing ovine vascular cell adhesion molecule-1 (VCAM-1). The mAb were raised against sheep umbilical vein endothelial cells (ShUVEC) and flow cytometric analysis demonstrated that one mAb, QE4G9, was cross-reactive with human VCAM-1 expressed on Chinese hamster ovary cell transfectants. Protein modulation studies on ShUVEC and immunoperoxidase staining of inflamed renal tissue further indicated the reactivity of the other two ovine mAb, QE1F3 and QE2G4, with ovine VCAM-1. The flow cytometric profile of the three mAb on stimulated ShUVEC was identical to that observed with the cross-reactive anti-human VCAM- mAb, HAE2-1. Peak expression occurred between 6-12 h after stimulation, followed by a slight decrease to a plateau extending beyond 48 h. In functional assays, all mAb inhibited adhesion of PMA-activated sheep PBMC to stimulated ShUVEC. In addition, QE4G9 inhibited proliferation of sheep PBMC in the mixed lymphocyte-endothelial cell reaction (MLER) by 56%. The results demonstrate that the three anti-ovine VCAM-1 mAb recognize functional epitopes on sheep vascular endothelial cells. These mAb will be valuable tools in the investigation of VCAM-1 expression in various pathophysiological conditions using sheep models, and in the study of VCAM-1-mediated leucocyte-endothelial cell interactions, both in vitro and in vivo.