Effects of dietary supplementation with Bacillus spp. on immune response, intestinal health outcomes, and the nasal, otic, and fecal microbiota of healthy adult dogs

Wilson, Sofia Marie

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https://hdl.handle.net/2142/129536 Copy

Description

Title

Effects of dietary supplementation with Bacillus spp. on immune response, intestinal health outcomes, and the nasal, otic, and fecal microbiota of healthy adult dogs

Author(s)

Wilson, Sofia Marie

Issue Date

2025-04-21

Director of Research (if dissertation) or Advisor (if thesis)

Swanson, Kelly S

Doctoral Committee Chair(s)

Swanson, Kelly S

Committee Member(s)

• Fahey, Jr., George C.
• Miller, Michael J
• Loman, Brett R

Department of Study

Animal Sciences

Discipline

Animal Sciences

Degree Granting Institution

University of Illinois Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• canine microbiota
• probiotic

Abstract

Dietary supplementation with “biotics,” which include probiotics, prebiotics, synbiotics, and postbiotics, is a strategy used to modify the intestinal microbiota and promote host health. Although biotics are commonly incorporated into pet products, questions remain regarding appropriate biotic selection, mechanisms of action, optimum inclusion levels, and safety. Bacillus spp. are spore-forming bacteria able to survive in extreme conditions, such as the acidic stomach, and vegetative cells exhibit high metabolic activity associated with production of enzymes and antimicrobial compounds. However, questions remain regarding the mechanisms by which Bacillus spores and vegetative cells exert their benefits. Therefore, the purpose of this dissertation was to evaluate the probiotic potential of select Bacillus strains and their effects on the gut microbiome of dogs and assess their role in supporting gut health and immunity. The first aim was to determine the apparent total tract macronutrient digestibility (ATTD) of diets supplemented with fibers or biotics and to evaluate their effects on the fecal characteristics, metabolites, microbiota, and immunoglobulin (Ig) A concentrations of dogs. Adult female Beagle dogs (n=12; age=6.2 ± 1.6 yr; body weight=9.5 ± 1.1 kg) were used in a replicated 3×3 Latin square design to test three treatments: 1) control diet based on rice, chicken meal, tapioca starch, and cellulose + a placebo treat (CT); 2) diet based on rice, chicken meal, garbanzo beans, and cellulose + a placebo treat (GB); 3) diet based on rice, chicken meal, garbanzo beans, and a functional fiber/prebiotic blend  + a probiotic-containing treat (GBPP). The probiotic treat contained Bacillus subtilis and Bacillus amyloliquefaciens [2 × 109 colony-forming units (CFU)/d]. In each 28-day period, a 22-day diet adaptation was followed by a 5-day fecal collection phase. ATTD of dry matter (DM), organic matter, and energy were lower (P<0.001) and DM fecal output was higher (P<0.01) in dogs fed GBPP than CT or GB, whereas ATTD of crude protein was higher (P<0.001) in dogs fed CT and GBPP than GB. ATTD of fat was higher (P<0.001) and wet fecal output was lower (P<0.01) in dogs fed CT than GB or GBPP. Fecal DM% was higher (P<0.001) in dogs fed CT than GBPP or GB, and higher in dogs fed GBPP than GB. Fecal short-chain fatty acid concentrations were higher (P<0.001) in dogs fed GB than CT or GBPP, and higher in dogs fed GB than GBPP. Fecal IgA concentrations were higher (P<0.01) in dogs fed GB than CT. Fecal microbiota populations were affected by diet, with alpha diversity being higher (P<0.01) in dogs fed GB than CT, and beta diversity shifting following dietary fiber and biotic supplementation. The relative abundance of 24 bacterial genera were altered in dogs fed GB or GBPP than CT. Finally, serum triglyceride concentrations were lower in dogs fed GB than GBPP or CT. The second aim was to determine the effects of Bacillus coagulans GBI-30, 6086 on ATTD and the hematology, Ig concentrations, and fecal characteristics, metabolites, and microbiota populations of healthy adult dogs. Adult English Pointer dogs (n=12; age=5.9±2.5 yr; body weight=26.6±6.1 kg) were fed the same diet, but supplemented with B. coagulans or a placebo via gelatin capsules in a replicated 3×3 Latin square design. Capsules were administered daily before each feeding, with the following treatments tested: 1) basal diet + placebo (control; 250 mg maltodextrin); 2) basal diet + B. coagulans (low dose; 5 × 108 colony-forming units CFU/d); and 3) basal diet + B. coagulans (high dose; 2.5 × 109 CFU/d). In each 28-day period, a 22-day diet adaptation was followed by a 5-day sample collection phase. B. coagulans supplementation did not affect ATTD, food intake, fecal metabolites, immunoglobulin concentrations, or hematology, but did lower fecal scores (P<0.05; firmer stools). Using qPCR, fecal Faecalibacterium spp. abundance was greater (P<0.05) and fecal Bacteroides spp., Bifidobacterium spp., and Ruminococcus gnavus abundances tended to be greater (P<0.10) in dogs fed the low B. coagulans dose than those fed the placebo. Using 16S rRNA sequencing, the relative abundance of nasal Staphylococcus spp. tended to differ (P<0.10) between dogs supplemented with the low and high dose of B. coagulans. Within the pinnae, the relative abundances of Bifidobacterium spp. and Dubosiella spp. were greater (P<0.05) in dogs fed the low dose of B. coagulans than controls, while Allobaculum stercoricanis and Sutterella spp. abundances were lower (P<0.05) in dogs supplemented with the high dose of B. coagulans than controls. The third aim was to determine the effects of B. coagulans GBI-30, 6086 on the fecal scores, pH, DM percentage, and microbiota populations of dogs following an abrupt diet change. Adult English Pointer dogs (n=12; age=5.9±2.5 yr; body weight=26.6±6.1 kg) were used in a replicated 3×3 Latin square design and fed commercial diets containing no probiotics or prebiotics. The following treatments were administered orally in gelatin capsules before each daily feeding: 1) placebo control (250 mg maltodextrin/day); 2) B. coagulans (low dose; 5 × 108 CFU/day); and 3) B. coagulans (high dose; 2.5 × 109 CFU/day). An extruded kibble diet was fed for 28 days. Dogs were then abruptly switched to a canned diet and fed for 14 days, with fecal samples collected before and 2, 6, 10, and 14 days after diet change. The abrupt diet change reduced (P<0.0001) fecal DM content, increased (P<0.0001) fecal scores and pH, and reduced (P<0.0001) fecal bacterial species richness and phylogenetic diversity. Diet change also increased (P<0.001) fecal Bacteroidota, Fusobacteriota, and Proteobacteria, decreased (P<0.001) fecal Firmicutes, and altered about 40 fecal bacterial genera relative abundances. Diet-induced changes were not greatly impacted by B. coagulans, but fecal scores tended to be lower (i.e., firmer stools; P<0.10), fecal E. coli and Faecalibacterium abundances were greater (P<0.05), and fecal bacterial phylogenic diversity was higher (P<0.05) in dogs supplemented with the low dose than placebo-supplemented dogs. The final aim was to evaluate the fecal characteristics, microbiota populations, fecal IgA and calprotectin concentrations, as well as serum chemistry, hematology, and immune function of dogs supplemented with B. subtilis and B. amyloliquefaciens. Adult English Pointer dogs (n=20; age=5.0 ± 2.3 yr; BW=24.4 ± 3.8 kg) were used in a double-blind, placebo-controlled crossover design. Dogs were allotted to the placebo (n = 10; 3-4 g/d of maltodextrin) or probiotic group (n = 10; 3-4 g/d of probiotic powder, 2 × 108 CFU/g) and fed to maintain BW for 28 days. Fecal scores tended to be higher (P<0.10; i.e., looser stools) and fecal DM content was lower (P<0.05) in dogs consuming the probiotic than those consuming the placebo, but fecal IgA and calprotectin concentrations did not differ between treatments. Serum metabolites and hematology data were all within normal reference ranges, although eosinophils (counts and % of total white blood cells) were lower (P<0.05) in probiotic-supplemented dogs than those in the control group. Immune cells isolated from probiotic-supplemented dogs produced greater (P<0.05) tumor necrosis factor-alpha (TNF-α) than those from control dogs when cells were stimulated with zymosan, an agonist of toll-like receptor 2. Concentrations of serum lipopolysaccharide-binding protein (LBP), interleukin (IL)-1β, and IL-6 did not differ between treatment groups, however serum C-reactive protein (CRP) concentrations were higher (P<0.05) in dogs supplemented with the probiotic than those in the control group. Fecal microbial composition, evaluated by alpha and beta diversity indices, was not significantly impacted by probiotic supplementation, although the Shannon Diversity Index tended (P<0.10) to be higher in placebo-supplemented dogs. The relative abundances of fecal microbiota measured by 16S rRNA sequencing were not greatly affected by probiotic supplementation, although the relative abundance of fecal Blautia was lower (P<0.05) in dogs supplemented with the probiotic than those fed the placebo.  Our findings indicate that while B. coagulans, B. subtilis, and B. amyloliquefaciens can be safely supplemented to healthy dogs, their probiotic effects on GI health and immunity appear to be dose, species, or strain dependent. B. coagulans minimally impacted immune parameters, with a greater impact on stool quality and fecal microbial composition when dogs were fed a low dose of (5 × 108 CFU/d). However, few benefits were observed in dogs consuming the high dose of B. coagulans (2.5 × 109 CFU/d). Conversely, B. subtilis and B. amyloliquefaciens provided limited intestinal health benefits and reduced stool quality, although spores modulated innate immune responses in healthy dogs. Bacillus inclusion in canine diets did not substantially impact nutrient digestibility, whereas legume-based dietary fibers, with or without prebiotics and probiotics, were shown to reduce ATTD, and abruptly transitioning dogs from a kibble to canned diet negatively influenced fecal characteristics. Overall, diet and macronutrient composition were shown to influence fecal characteristics and drastically shift the fecal microbiota, regardless of Bacillus supplementation. Therefore, the probiotic potential of Bacillus spp. may be enhanced if supplemented in combination with prebiotics or as a multi-strain Bacillus probiotic mixture to offer complementary benefits to gut health and immunity by providing support for a more diverse range of microbes.

Graduation Semester

2025-05

Type of Resource

Thesis

Handle URL

https://hdl.handle.net/2142/129536

Copyright and License Information

Copyright 2025 Sofia M. Wilson

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