Twelve ruminally cannulated Holstein calves (age, 12 ± 3 weeks) were used to identify the effect of a probiotic comprised of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum on ruminal components. The calves were adapted to a diet containing a 50% high-concentrate (standard diet) for 1 week, and then, the probiotic was given once daily for 5 days (day 1–5) at 1.5 or 3.0 g/100 kg body weight to groups of four calves each. Four additional calves fed the standard diet without probiotic served as the corresponding control. Ruminal pH was measured continuously throughout the 15-day experimental period. Ruminal fluid was collected via a fistula at a defined time predose and on days 7 and 14 to assess volatile fatty acid (VFA), lactic acid and ammonia-nitrogen concentrations, as well as the bacterial community. The probiotic at either dose improved the reduced 24-hr mean ruminal pH in calves. The circadian patterns of the 1 hr mean ruminal pH were identical between the probiotic doses. In both probiotic groups, ruminal lactic acid concentrations remained significantly lower than that of the control. Probiotic did not affect ruminal VFA concentrations. L. plantarum and C. butyricum were not detected in the rumen of calves given the high-dose probiotic, whereas Enterococcus spp. remained unchanged. These results suggest that calves given a probiotic had stable ruminal pH levels (6.6–6.8), presumably due to the effects of the probiotic on stabilizing rumen-predominant bacteria, which consume greater lactate in the rumen.

Subpopulations of peripheral leukocytes and cytokine mRNA expression levels were evaluated in scouring and healthy Holstein calves (age 10 ± 5 days; n=42) treated with a probiotic consisting of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum. The calves were assigned to the scouring or healthy group and then subdivided into pathogen-positive treated (n=8), pathogen-positive control (n=8), pathogen-negative treated (n=6), pathogen-negative control (n=6), healthy treated (n=6) and healthy control (n=8) groups. A single dose of the probiotic (3.0 g/100 kg body weight) was given to each calf in the treatment groups for 5 days. Blood samples were collected on the first day of scour occurrence (day 0) and on day 7. In the scouring calves, smaller peripheral leukocyte subpopulations and cytokine mRNA expression levels were noted on day 0. The numbers of CD3+ T cells; CD4+, CD8+ and WC1+ γδ T cell subsets; and CD14+, CD21+ and CD282+ (TLR2) cells were significantly increased in the scouring and healthy treated calves on day 7. Furthermore, interleukin-6, tumor necrosis factor-alpha and interferon-gamma mRNA expression was elevated in the peripheral leukocytes of the scouring and healthy treated calves on day 7. The scouring calves given the probiotic recovered on day 7. A significantly smaller number of peripheral leukocytes and lower cytokine mRNA expression level might be induced by scouring in calves. Repeated probiotic administration might stimulate cellular immunity and encourage recovery from scouring in pre-weaning Holstein calves.

Eight Holstein calves (10 ± 3 weeks) were used to examine the interaction between a bacteria-based probiotic agent (probiotic) and the function of peripheral blood mononuclear cells (PBMCs). The probiotic, consisting of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum, was administered orally at 3.0 g/100 kg body weight to calves once daily for 5 consecutive days. Calves given the vehicle alone with no probiotic served as the control. In the treatment group, increases in numbers of CD282+ (TLR2) monocytes, CD3+ T cells and CD4+, CD8+ and WC1+ γδ T cell subsets were noted on day 7 post-placement compared to predose day and the control group. Expression of interleukin (IL)-6, interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α) was elevated in peripheral leukocytes on days 7 and 14. These results suggest that peripheral blood leukocytes in healthy calves may be stimulated via the gastrointestinal microbiota, which was increased by the oral probiotic treatment, with overall stability of the rumen bacterial flora. The 5-day repeated administration of a bacteria-based probiotic may enhance cellular immune function in weaned calves.

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