When milk-fed mice were orally inoculated with Clostridium ramosum C1, this strain proliferated in the gut and became the dominant component of the fecal microflora. In this experimental model, bovine lactoferrin (bLF) administered with milk suppressed the proliferation of this strain in vivo and decreased the numbers of C. ramosum and other bacteria in the feces. This bacteriostatic effect of bLF was dependent on the concentration of bLF, the duration of feeding, and the administered dose of C. ramosum C1. Compared with bovine serum albumin, ovalbumin, bovine whey protein isolate, or bovine casein, only bLF showed this specific activity. A similar effect of bLF was observed after oral inoculation with C. ramosum JCM 1298, C. paraputrificum VPI 6372, or C. perfringens ATCC 13124. A hydrolysate prepared by digestion of bLF with porcine pepsin showed the same inhibitory effect on proliferation of C. ramosum in vivo as occurred with undigested bLF. These results indicate that ingested bLF can exert a bacteriostatic effect against clostridia in the gut even after it has been digested to some extent.

Various investigators have discussed floral microorganisms influence on a host's immune system (4, 6, 9). Most of these statements come from investigations comparing responses of germfree animals and flora-bearing ones. The present authors attempted to analyze the effect of an indigenous floral organisms on the host under a more simplified condition. Bifidobacterium longum (BL) was given per os to germfree mice (GF) of BALB/c background and various aspects of immune responses to the associated organism were investigated first; antibody (mainly IgA) and cell-mediated immune (CMI) responses to BL, translocations of BL into the internal organs, and shift of lymphocyte subsets in the lymphoid organs were found following per os administration of BL. Some activities of BL on in vitro cultured lymphocytes were also found. Additionally, it was shown that the BL-monoassociated mouse (GB) exhibits beneficial effects in respect to specific immune response and non-specific antibacterial defence reactions to unrelated antigenic substances. These non-specific effects were seen on lethal activity of Escherichia coliendotoxin, IgA production to cholera toxin (CT), and induction of oral tolerance to unrelated antigens. IMMUNOLOGICAL AND MICROBIAL CHANGES AFTER MONOASSOCIATION WITH BL

A cell wall preparation (whole peptidoglycan, WPG) derived from Bifidobacterium infantis exhibited antitumor activity when WPG and Meth A cells were mixed and injected subcutaneously (suppression test) in flora-bearing (FB) BALB/c mice. This antitumor activity was markedly reduced when the germfree (GF) BALB/c mice were used as a host. Histological examination of the injected site revealed vigorous polymorphonuclear leukocyte (PMN) infiltration and tumor cell degeneration at day 1 to 3 postimplantation of WPGMeth A mixture in both FB and GF mice, but later on re-appearance of tumor cell mass was seen in many GF but in only a few FB mice. Similar but somewhat weaker suppressive effects of WPG were seen in FB athymic nu/nu mice in which severe PMN infiltration was also developed. Results indicated that the early inflammatory reaction composed mostly of PMN played a role as the major effector mechanisms in the suppression test, and inferior antitumor activity of PMN from GF mice to that from FB mice was implicated. However, since FB euthymic but not FB nu/nu mice showed development of antitumor immunity in re-implantation, involvement of immunological capacity might have played an additional role in the suppression efficiency.