The aim of the present study was to examine the effects of oral supplementation of newborn Balb/c mice with bifidobacteria (B. infantis, B. bifidum) and iron-free apo-lactoferrin (bovine, human) on gut endotoxin concentration and mucosal immunity. Endotoxin concentration was measured in ileocecal filtrates at 7, 14, 21, and 28 days postdelivery by a quantitative limulus amebocyte lysate test. While endotoxin levels in bifidobacteria-fed mice showed a steady rise over time, they were consistently lower than that observed in control animals. Results of lactoferrin supplementation varied depending on the specific time point, but overall by day 28, all treatment groups showed lower intestinal endotoxin concentrations compared to saline fed animals. Neither bifidobacteria nor lactoferrin stimulated an increase in B or T cells, or in cytokine production (IL-6, TNF-alpha, INF-gamma), in Peyer's patches as measured by flow cytometry. Bifidobacteria and lactoferrin were well tolerated as dietary supplements and showed promising potential to reduce gut endotoxin levels.

Different hypervariable (V) regions of the archaeal 16S rRNA gene (rrs) were compared systematically to establish a preferred V region(s) for use in Archaea-specific PCR-denaturing gradient gel electrophoresis (DGGE). The PCR products of the V3 region produced the most informative DGGE profiles and permitted identification of common methanogens from rumen samples from sheep. This study also showed that different methanogens might be detected when different V regions are targeted by PCR-DGGE. Dietary fat appeared to transiently stimulate Methanosphaera stadtmanae but inhibit Methanobrevibacter sp. strain AbM4 in rumen samples.Archaea can thrive in various natural and engineered environments, and many of them can grow in habitats at the extreme limits (in terms of temperature, pH, salinity, anaerobiosis, etc.) of conditions that allow life on earth (7a). From a pragmatic perspective, archaeal extremophiles can be exploited for biotechnological applications (9a, 25a), while methanogens mediate the terminal step of the carbon cycle (through methanogenesis) in anaerobic environments, including rumens and anaerobic digestors (2b, 20a). The methanogens present in the latter two habitats have recently received renewed interest because it has been recognized that ruminal methanogens contribute significantly to emission of methane (a potent greenhouse gas) and to loss of feed energy (22a), while the methanogens present in anaerobic digestors produce methane biogas as a valuable bioenergy source from a variety of types of biomass (2a). Since Archaea are typically fastidious organisms that are difficult to culture in the laboratory, they are often analyzed by molecular biology techniques (1, 28).Community profiling by PCR-denaturing gradient gel electrophoresis (DGGE) has been widely used to examine both temporal succession in microbial communities and spatial variations in microbial diversity in the same type of microbial communities (21, 24). It is especially useful for identifying samples of interest for in-depth analyses in a large number of samples (9,13,16,20). However, even using universal primers, Archaea were probably not accounted for in such studies because sequencing of excised DGGE bands rarely yielded Archaea-like sequences (13,14). This is probably attributable to the low abundance of Archaea in most microbial communities and/or primer mismatch. Thus, Archaea-targeting PCR-DGGE has been used increasingly for profiling Archaea in various environmental samples (1,17,27,33). A recent study also reported profiling of ruminal methanogens by PCR-DGGE, but no primer was reported (8). In all these studies different primer sets were used and different hypervariable (V) regions were targeted, but no justification or rationale was presented. In a previous study we noted that different V regions of the bacterial 16S rRNA gene (rrs) can produce different DGGE patterns (number and separation of DGGE bands) (31). We hypothesized that this is also true for Archaea-specific PCR-DGGE. In this study we first evaluated a battery...

The regulation of bovine mammary lactoferrin, an important component of the antimicrobial defenses of the mammary gland, is poorly understood compared with the other milk proteins. The complete sequence for bovine lactoferrin mRNA shows it to be highly homologous to other lactoferrins and transferrins. However, regional differences in the deduced AA sequence of bovine lactoferrin compared with human lactoferrin and transferrin imply functional differences between them. Steady-state levels of bovine lactoferrin mRNA (by Northern blot) in the bovine mammary gland indicate that bovine lactoferrin expression is minimal in the developing and lactating gland but is strongly induced by mammary involution. The overall regulation of bovine lactoferrin in the mammary gland appears to be contrary to that of the other milk proteins. Features identified in the mRNA of bovine mammary lactoferrin may contribute to the differences in regulation between lactoferrin and other bovine milk proteins and to differences in concentrations of lactoferrin in milk across species. Lactoferrin secretion by bovine mammary cells grown in vitro does not appear to be dependent on prolactin and shows regulation by substratum, serum, and cell population to be different from that for casein. In contrast to casein, efficient secretion of lactoferrin from the cell does not require detachment of collagen substratum.

Electroimmunodiffusion assay was used to quantitate changes in lactoferrin concentration in mammary secretions during involution of the bovine mammary gland. Concentration of lactoferrin began to increase 2 to 4 days after cessation of regular milking and continued to increase linearly at a rate of 1.15 mg/ml per day as a result of increased net synthesis of lactoferrin during the first 14 to 21 days of involution. Maximum lactoferrin concentration (approximately 20 mg/ml) was attained after 3 to 4 wk of involution. These changes represent a 100-fold increase in lactoferrin concentration over that in normal milk. Maximum lactoferrin concentration was variable between cows. In some cows, the concentration of lactoferrin plateaued at less than 10 mg/ml after 10 days of involution. In others, much higher lactoferrin concentrations of 75 to 100 mg/ml were measured. Lactoferrin concentration decreased markedly prior to parturition and onset of lactation. The increase in lactoferrin concentration during mammary gland involution appeared to be related closely to the process of involution.

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