Comparative studies of microbial populations in the rumen, duodenum, ileum and faeces of lactating dairy cows.

AIMS: Understanding factors that influence the composition of microbial populations of the digestive system of dairy cattle will be key in regulating these populations to improve animal performance. Although rumen microbes are well studied, little is known of the dynamics and role of microbial populations in the small intestine of cows. Comparisons of fingerprints of microbial populations were used to investigate the effects of gastrointestinal (GI) segment and animal on community structure. METHODS AND RESULTS: Samples from four lactating dairy cows with ruminal, duodenal and ileal cannulae were collected. Terminal-restriction fragment length polymorphism (T-RFLP) comparisons of small subunit rRNA genes revealed differences in microbial populations between GI segments (P < 0.05). No significant differences in either methanogen populations or microbial community profiles between animals were observed. Quantitative PCR was used to assay relative changes in methanogen numbers compared to procaryote rRNA gene numbers, and direct microscopic counts were used to enumerate total procaryote numbers of the duodenal and ileal samples. CONCLUSIONS: T-RFLP comparisons illustrate significant changes in microbial diversity as digesta passes from one segment to another. Direct counts indicate that microbial numbers are reduced by eight orders of magnitude from the rumen, through the abomasum, and into the duodenum (from c. 10(12) to c. 3.6 x 10(4) cells per ml). Quantitative PCR analyses of rRNA genes indicate that methanogens are present in the duodenum and ileum. SIGNIFICANCE AND IMPACT OF THE STUDY: The contribution of microbial populations of the small intestine to the nutrition and health of cattle is seldom addressed but warrants further investigation.

Intimal plaque development and oxidative stress in cholesterol-induced atherosclerosis in New Zealand rabbits.

Although oxidative stress is well known in atherogenesis, the origin, nature and kinetics of free radicals involved have not been well described till now. Here, we correlated parameters of oxidative stress with cellular components during induction and stabilization of aortic intimal lesions which were induced in rabbits by feeding a cholesterol-enriched diet for 6 weeks and a normal diet for further 68 weeks. Plasma lipids, aortic plaque size and composition (macrophages, smooth muscle cells, oxidized LDL by morphometry), as well as aortic radical production (by luminol-enhanced chemiluminescence and TEMPO-9AC fluorescence) were measured after various time points. The parameters of oxidative stress were correlated with the different cellular components of the aortic plaques. The plaques increased until week 21, no significant regression was found until week 74, plasma cholesterol was maximal at week 6. Macrophages, oxidized LDL and generation of different species of free radicals were increased during plaque development, yet with different time kinetics. Whereas chemiluminescence correlated only weakly with the amount of intimal macrophages, strong correlations were found between TEMPO fluorescence and smooth muscle cells (r = 0.4778, P < 0.001) and between macrophages and oxidized LDL (r = 0.5896, P < 0.0001). Different indicators of oxidative stress were increased during plaque progression and stabilization. However, the various correlations show, that distinct types of reactive species secreted probably from macrophages and smooth muscle cells contribute to oxidative stress in the different phases of plaque development.

Phage-displayed peptides as biosensor reagents.

This study investigated the potential to utilize phage-displayed peptides as reagents in sensor applications. A library of random 12-mers displayed on phage was panned against staphylococcal enterotoxin B (SEB), a causative agent of food poisoning. Nine SEB binding phage clones were isolated, all of which share the consensus sequence Trp His Lys at their amino terminus. Binding of several of these phage was shown to be inhibited when they were assayed in a competitive enzyme-linked immunosorbent assay (ELISA) format with synthesized peptide corresponding to the peptide-encoding region of one of the clones. Whole phage were labeled with the dye Cy5, and incorporated into fluoroimmunoassays. Labeled phage were able to detect SEB down to a concentration of 1.4 ng/well in a fluorescence-based immunoassay. When incorporated into an automated fluorescence-based sensing assay, Cy5-labeled phage bound to probes coated with SEB generated a robust signal of about 10,000 pA, vs a signal of 1,000 pA using a control fiber coated with streptavidin. These results demonstrate the potential for development of phage-based sensor reagents.