Effects of combined Bacillus subtilis and Bacillus amyloliquefaciens supplementation on fecal characteristics, fecal microbiota, and immune function of healthy adult dogs

Bacillus species are spore-forming bacteria with characteristics that support their use as probiotics; however, the effects of combined Bacillus subtilis and Bacillus amyloliquefaciens supplementation on intestinal and immune outcomes have been minimally studied in dogs. This study evaluated fecal characteristics, microbiota, fecal immunoglobulin A and calprotectin concentrations, serum chemistry, hematology, and immune function in healthy adult dogs supplemented with B. subtilis and B. amyloliquefaciens . Twenty healthy adult English Pointer dogs (age = 5.0 ± 2.3 yr.; BW = 24.4 ± 3.8 kg) were enrolled in a randomized, double-blinded, placebo-controlled crossover study. Following a 21-d adaptation period, dogs were randomly assigned to one of two treatment sequences. During the first 28-d experimental period, 10 dogs received a placebo (maltodextrin) and 10 received a probiotic containing B. subtilis and B. amyloliquefaciens (2 × 10 8 CFU/g). Treatments were top-dressed on the diet at 3–4 g/d. After a 21-d washout, dogs crossed over to the opposite treatment for a second 28-d period. Data were analyzed using the MIXED procedure of SAS 9.4, with p < 0.05 considered significant and p < 0.10 considered a trend. Fecal scores tended to be higher ( p < 0.10; looser stools), and fecal dry matter was lower ( p < 0.05), in probiotic-supplemented dogs than controls, whereas fecal IgA and calprotectin concentrations were unaffected. Serum chemistry and hematology values were within normal reference ranges, although eosinophil counts and percentages were lower ( p < 0.05) in probiotic-supplemented dogs. Peripheral blood mononuclear cells from probiotic-supplemented dogs produced greater TNF-α concentrations following stimulation with zymosan, a TLR2 agonist ( p < 0.01). Serum LBP, IL-1β, and IL-6 concentrations were unaffected, whereas serum CRP was higher ( p < 0.05) in probiotic-supplemented dogs. Fecal microbial composition was largely unaffected, although Shannon diversity tended to be higher ( p < 0.10) in controls and the relative abundance of Blautia was lower ( p < 0.05) in probiotic-supplemented dogs. Overall, supplementation provided limited intestinal health benefits, while treatment differences in TLR2-stimulated TNF-α production and serum CRP suggest effects on selected innate immune-related outcomes that warrant further investigation.