Safety and Tolerance of Dietary Supplementation With a Canine-Derived Probiotic (Bifidobacterium animalis Strain AHC7) Fed to Growing Dogs

Abstract

Although probiotics are generally considered to be safe, their increasingly widespread use warrants better understanding of their risks in companion animals. This study evaluated the safety and tolerance of dietary supplementation with a canine-derived probiotic, Bifidobacterium animalis strain AHC7 (Prostora, Procter & Gamble Pet Care), fed to growing beagles beginning at approximately 6 months of age (11 males; 9 females). Probiotic B. animalis AHC7 administered orally once per day at a dose of up to 5 x 1010 colony-forming units for at least 12 consecutive weeks was well tolerated with no safety concerns.

Full text

Safety and Tolerance of Dietary Supplementation
With a Canine-Derived Probiotic (Bifidobacterium
animalis Strain AHC7) Fed to Growing Dogs*
R. L. Kelley, MSa,**
Jean Soon Park, PhDa
Liam O’Mahony, PhDb,†
Debbie Minikhiem, MSa
Andrew Fix, DVM, PhDa
R. L. Kelley, J. Soon Park, L. O’Mahony, D. Minikhiem, and A. Fix
E1
CLINICAL RELEVANCE
Although probiotics are generally considered to be safe, their increasingly wide-
spread use warrants better understanding of their risks in companion animals.
This study evaluated the safety and tolerance of dietary supplementation with a
canine-derived probiotic, Bifidobacterium animalis strain AHC7 (Prostora, Proc-
ter & Gamble Pet Care), fed to growing beagles beginning at approximately 6
months of age (11 males; 9 females). Probiotic B. animalis AHC7 administered
orally once per day at a dose of up to 5 ×1010 colony-forming units for at least
12 consecutive weeks was well tolerated with no safety concerns.
©Copyright 2010 MediMedia Animal Health. This document is for internal purposes only.
Reprinting or posting on an external website without written permission from MMAH is a violation of copyright laws.
*This study was funded by Procter & Gamble Pet Care, Lewisburg, OH.
**Correspondence should be sent to R. L. Kelley: phone, 937-962-7296; fax, 513-277-7921; e-mail, kelley.
rl@pg.com.
†Dr. O’Mahony’s current address is Swiss Institute for Allergy and Asthma Research, University of Zurich,
Obere Strasse 22, CH-7270 Davos, Switzerland.
■INTRODUCTION
Probiotics are live microorganisms that con-
fer a health benefit on the host when adminis-
tered in adequate amounts. Combinations of
microorganisms, particularly species of lactic
acid bacteria, including Lactobacillus and Lac-
tococcus, have traditionally been used in fer-
mented dairy products to promote human
health through their influence on the micro-
bial ecology of the host and the incidence of
diarrhea.1Probiotics have also been used for
many years in animal husbandry and have
been demonstrated to be effective in improv-
ing gastrointestinal health. However, in the
case of companion animals, particularly dogs
and cats, less is known about the potential ap-
plications of probiotics.
Despite the fact that few studies have been
conducted examining the efficacy and safety of
probiotic supplementation in companion ani-
aProcter & Gamble Pet Care
Technical Center
6571 State Route 503 North
Lewisburg, OH 45338
bAlimentary Health Ltd
2800 Cork Airport Business Park
Kinsale Road, Cork
Ireland

Veterinary Therapeutics • Vol. 11, No. 3, Fall 2010
E2
risks in companion animals is
warranted.
A strain of Bifidobacterium (Bi-
fidobacterium animalis AHC7)
was recently isolated from healthy
canine gastrointestinal tissue and
tested in adult dogs as a potential
probiotic.8In this study, dogs
received 1.5 ×109colony-form-
ing units (CFU)/day of B. ani-
malis AHC7 for 6 weeks with
no health concerns. A subsequent
study found that supplementa-
tion with B. animalis AHC7 was
effective in reducing the time to
resolution of acute idiopathic di-
arrhea when fed at 2 ×1010
CFU/day for a maximum of 2
weeks with no health concerns.9
The purpose of the present study
was to evaluate the safety and tol-
erance of B. animalis AHC7
when administered at a dose of
up to 5 ×1010 CFU/day for a
longer duration (at least 12 con-
secutive weeks) to growing dogs.
■MATERIALS AND
METHODS
Twenty beagles from four lit-
ters were enrolled in the study at
the Procter & Gamble Pet
Health Nutrition Center (PHNC; Lewisburg,
OH). The average age of the dogs at the time
of enrollment was approximately 6 months.
Before the study began, all dogs received a
complete physical examination by a veterinari-
an, and blood was collected for evaluation of
hematologic and serum chemistry parameters.
All dogs were found to be in good health with
no medical conditions that would adversely af-
fect the study. All animal procedures met US
Department of Agriculture procedures for lab-
mals, the number of commercially available
probiotic products has proliferated rapidly.
This has led to concern about the quality,
labeling, and verification of claims attributed
to some of these products.2Although probi-
otics are generally considered to be safe in hu-
mans,3–6 it has been recommended that their
use in severely compromised human patients
be avoided.7Given their increasingly wide-
spread use for clinical management in un-
healthy pets, a better understanding of their
Figure 1. Body weight increased gradually over the course of the study in
both supplement groups as expected, and no statistically significant differ-
ences were noted between the groups at any time point.
Week
12
11
10
9
8
7
6
5
4
012345678910 11 12 13 14 15 16
Body weight (kg)
Low concentration
High concentration
Least Squares Mean Body
Weight (kg) at Each Time Point
Figure 2. Weekly food intake did not change over the course of the study
in either supplement group, and no statistically significant differences
were noted between the groups at any time point.
Week
2.5
2
1.5
1
0.5
0
012345678910 11 12 13 14 15 16
Weekly food intake (kg)
Low concentration
High concentration
Least Squares Mean Weekly
Food Intake (kg) at Each Time Point

E3
R. L. Kelley, J. Soon Park, L. O’Mahony, D. Minikhiem, and A. Fix
oratory care and were approved
by the Procter & Gamble Pet
Care Institutional Animal Care
and Use Committee.
The study began with a 25-day
prefeeding period in which all
dogs were fed a commercial dry
diet (Eukanuba Medium Breed
Puppy, Procter & Gamble Pet
Care) twice daily without active
or placebo supplements, followed
by a 7-day acclimation period in
which all dogs received placebo
supplements (0 CFU) in addition
to daily rations of the commercial
diet. Active and placebo supple-
ments were provided in the form
of cocoa butter treats. Each meal
contained half of the estimated
caloric need, with daily caloric in-
take adjusted to help maintain an optimal
body condition. At the end of the acclimation
period (baseline, week 0), dogs were weighed,
rated for body condition score (BCS), and ran-
domly assigned within each litter to one of two
active supplements containing B. animalis
AHC7 (high or low concentration) after bal-
ancing for sex, weight, and BCS by two
PHNC personnel trained in the scoring proce-
dure. The BCS was rated on a 5-point scale
with half-points in between (1 = thin; 2 = un-
derweight; 3 = ideal; 4 = overweight; 5 =
obese). Active supplementation began the next
day and continued throughout the following
12-week period (days 1 through 84).
Active supplements were given once daily
with the morning meal for the duration of the
12-week supplementation period. Ten dogs (6
males; 4 females) received a low concentration
of B. animalis AHC7 (1 ×109CFU; overall av-
erage: 1.43 ± 0.44 ×109CFU), and 10 (5
males; 5 females) received a high concentration
of B. animalis AHC7 (5 ×1010 CFU; overall
average: 4.93 ± 0.70 ×1010 CFU). The supple-
mentation period was followed by a 4-week
observation period (days 85 through 112) in
which all dogs received placebo supplements (0
CFU) in addition to daily rations of the com-
mercial diet.
Analyses were performed on supplements
every 4 weeks during the study to ensure stable
levels of B. animalis strain AHC7. All B. ani-
malis strain AHC7 microbial populations were
determined in triplicate based on CFU plate
counts using selective growth media for Bifi-
dobacterium spp (de Man, Rogosa, Sharpe
[MRS] agar; Oxoid, Hampshire, UK).
Dogs were monitored daily for stool scores,
food intake, and daily activity. Stool scores were
rated on a five-point scale (1 = liquid with or
without particle matter; 2 = soft, shapeless;
3 = soft, with shape; 4 = firm, well formed; 5 =
extremely dry). The primary fecal score from
the first defecation of the day was analyzed. The
dogs were double-housed (cohabitating dogs
were in the same supplementation group);
Figure 3. Supplementation with Bifidobacterium animalis AHC7 sig-
nificantly increased the percentage of B. animalis AHC7 present in fecal
samples at weeks 6 and 12 in the high-concentration group and tended to
increase the percentage in the low-concentration group.
Time
3
2.5
2
1.5
1
0.5
0
Baseline Week 6 Week 12 Week 16
B. animalis AHC7 (% of total bacteria)
Low concentration
High concentration
Mean (± SE) Bifidobacterium animalis AHC7
Concentrations as a Percentage of the Total
Bacterial Concentrations at each Time Point
(text continues on page E8)

TABLE 1. Hematology Parameters of Dogs Randomly Assigned to Either a Low or a High Concentration of Probiotic Bifidobacterium
animalis AHC7a
Iams 18-
ANTECH Week-Old
Adult Puppy
Reference Reference Baseline (Week 0) Week 6 Week 12 Week 16
Range Range Low High Low High Low High Low High
Parameter 2009 2006 Concentration Concentration Concentration Concentration Concentration Concentration Concentration Concentration
WBC 4.0–15.5 3.35–70 8.49 8.67 7.88 7.77 7.61 7.95 7.84 7.62
(1000/µL) ± 0.53 ± 0.53 ± 0.53 ± 0.53 ± 0.53 ± 0.53 ± 0.53 ± 0.53
% Neut 60–77 28–89 61.98 63.85 59.82 64.57 61.94 64.43 63.42 66.44
± 1.93 ± 1.93 ± 1.93 ± 1.93 ± 1.93 ± 1.93 ± 1.93 ± 1.93
% Lymp 12–30 0–55 25.45 26.08 30.17 27.60 29.40 26.32 27.74 25.21
± 2.11 ± 2.11 ± 2.11 ± 2.11 ± 2.11 ± 2.11 ± 2.11 ± 2.11
% Monos 3–10 0–13 8.51 9.43 8.75 7.35 7.71 8.49 8.18 7.41
± 0.77 ± 0.77 ± 0.77 ± 0.77b± 0.77 ± 0.77 ± 0.77 ± 0.77b
% Eos 2–10 0–14 0.96 0.62 0.83 0.24 0.89 0.48 0.48 0.74
± 0.30 ± 0.30 ± 0.30 ± 0.30 ± 0.30 ± 0.30 ± 0.30 ± 0.30
% Baso 0–1 0–1 0.11 0.02 0.43 0.24 0.07 0.27 0.18 0.20
± 0.06 ±0.06 ± 0.06b,c ± 0.06b,c ± 0.06c± 0.06b,c ± 0.06 ± 0.06b
% Ban 0–3 0–1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00
RBC 4.8–9.3 4.85–6.55 6.35 6.46 6.99 6.89 6.89 7.04 7.19 7.35
(106/µL) ± 0.12 ± 0.12 ± 0.12b± 0.12b± 0.12b± 0.12b± 0.12b± 0.12b
Hgb 12.1–20.3 11.3–15.1 14.39 14.66 15.48 15.46 16.15 16.57 16.82 17.29
(g/dL) ± 0.29 ± 0.29 ± 0.29b± 0.29b± 0.29b± 0.29b± 0.29b± 0.29b
HCT (%) 36–60 35.2–52.2 44.31 44.78 48.31 47.60 49.25 50.47 51.31 52.58
± 0.85 ± 0.85 ± 0.85b± 0.85b± 0.85b± 0.85b± 0.85b± 0.85b
MCV (fL) 58–79 61–83.6 69.90 69.43 69.07 69.11 71.51 71.63 71.39 71.62b
± 0.53 ± 0.53 ± 0.53b± 0.53 ± 0.53b± 0.53b± 0.53b± 0.53b
Table continues on next page

MCH (pg) 19–28 21.7–25 22.69 22.74 22.15 22.45 23.47 23.53 23.42 23.55
± 0.20 ± 0.20 ± 0.20b± 0.20b± 0.20b± 0.20b± 0.20b± 0.20b
MCHC 30–38 21.6–33.9 32.45 32.73 32.08 32.48 32.83 32.85 32.77 32.88
(g/dL) ± 0.09c± 0.09c± 0.09b,c ± 0.09b,c ± 0.09b± 0.09 ± 0.09b± 0.09
PLT 170–400 130–429 296.60 330.10 287.50 292.00 301.90 311.50 312.60 310.80
(1000/µL) ± 17.96 ± 17.96 ± 17.96 ± 17.96b± 17.96 ± 17.96 ± 17.96 ± 17.9
NeutAb 2.06–10.60 1.91–19.6 5.30 5.57 4.75 5.06 4.74 5.14 4.99 5.10
(1000/µL) ± 0.43 ± 0.43 ± 0.43 ± 0.43 ± 0.43 ± 0.43 ± 0.43 ± 0.43
LymAb 0.69–4.50 0–7.26 2.20 2.24 2.35 2.09 2.25 2.07 2.17 1.89
(1000/µL) ± 0.20 ± 0.20 ± 0.20 ± 0.20 ± 0.20 ± 0.20 ± 0.20 ± 0.20
MonoAb 0–0.84 0–1.4 0.71 0.79 0.69 0.58 0.56 0.68 0.63 0.56
(1000/µL) ± 0.07 ± 0.07 ± 0.07 ± 0.07b± 0.07 ± 0.07 ± 0.07 ± 0.07b
EosAb 0–1.20 0–1.4 0.08 0.07 0.06 0.02 0.06 0.04 0.03 0.05
(1000/µL) ± 0.02 ±0.02 ± 0.02 ± 0.02 ± 0.02 ± 0.02 ± 0.02 ± 0.02
BasoAb 0–0.15 0 0.01 0.00 0.03 0.02 0.01 0.02 0.01 0.02
(1000/µL) ± 0.01 ±0.01 ± 0.01b,c ± 0.01c± 0.01 ± 0.01b± 0.01 ± 0.01
BanAb 0–0.30 0–0.5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
(1000/µL) ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00
aData shown are least squares mean ± SE.
bStatistically significant (P<.05) change from baseline within the supplement group based on a repeated measure, mixed model method with time point and supplement level as the fixed
factors.
cStatistically significant (P<.05) difference between the supplement groups based on a repeated measure, mixed model method with time point and supplement level as the fixed factors.
TABLE 1. Hematology Parameters of Dogs Randomly Assigned to Either a Low or a High Concentration of Probiotic Bifidobacterium
animalis AHC7a(cont.)
Iams 18-
ANTECH Week-Old
Adult Puppy
Reference Reference Baseline (Week 0) Week 6 Week 12 Week 16
Range Range Low High Low High Low High Low High
Parameter 2009 2006 Concentration Concentration Concentration Concentration Concentration Concentration Concentration Concentration

TABLE 2. Serum Chemistry Profile Parameters of Dogs Randomly Assigned to Either a Low or a High Concentration of Probiotic
Bifidobacterium animalis AHC7a
Iams 18-
ANTECH Week-Old
Adult Puppy
Reference Reference Baseline (Week 0) Week 6 Week 12 Week 16
Range Range Low High Low High Low High Low High
Parameter 2009 2006 Concentration Concentration Concentration Concentration Concentration Concentration Concentration Concentration
Chol 92–324 166–316 216.20 206.10 230.20 223.40 259.60 251.80 240.20 234.40
(mg/dL) ± 9.04 ± 9.04 ± 6.85b± 6.85b± 8.75b± 8.75b± 8.24b± 8.24b
Na 139–154 139–153 148.50 149.10 150.30 151.50 155.00 155.50 148.00 149.70
(mmol/L) ± 0.40 ± 0.40 ± 0.85b± 0.85b± 0.64b± 0.64b± 0.91 ± 0.91
K 3.6–5.5 4–6.2 4.32 4.41 4.45 4.34 4.50 4.48 4.29 4.20
(mmol/L) ± 0.09 ± 0.09 ± 0.09 ± 0.09 ± 0.10 ± 0.10 ± 0.08 ± 0.08
Cl 102–120 106–117 113.00 113.30 115.50 116.00 118.70 119.20 113.30 114.90
(mmol/L) ± 0.46 ± 0.46 ± 0.62b± 0.62b± 0.53b± 0.53b± 0.43c± 0.43b
TB 0.1–0.3 0.1–0.2 0.11 0.10 0.10 0.10 0.14 0.14 0.11 0.12
(mg/dL) ± 0.01 ± 0.01 ± 0.01 ± 0.01 ± 0.02 ± 0.02b± 0.02 ± 0.02
GGT 1–12 3–14 1.50 1.50 1.95 2.25 2.20 1.80 2.20 2.00
(U/L) ± 0.00 ± 0.00 ± 0.72 ± 1.30 ± 0.95 ± 0.63 ± 0.95 ± 1.15
ALT 12–118 24–338 41.40 40.10 57.40 53.60 42.30 43.20 41.10 44.20
(U/L) ± 3.93 ± 3.93 ± 8.91b± 8.91 ± 3.74 ± 3.74 ± 3.99 ± 3.99
AST 15–66 17–105 36.40 38.90 48.70 51.80 37.50 44.40 36.80 40.30
(U/L) ± 2.43 ± 2.43 ± 6.24b± 6.24 ± 3.51 ± 3.51b± 4.13 ± 4.13
LDH 20–500 30–685 122.40 126.70 93.40 95.10 129.10 152.00 122.40 108.30
(U/L) ± 21.13 ± 21.13 ± 9.18 ± 9.18b± 21.32 ± 21.32 ± 18.62 ± 18.62
Mg 1.5–2.5 0.3–3.2 2.25 2.34 2.32 2.29 2.29 2.23 2.20 2.17
(mEq/L) ± 0.07 ± 0.07 ± 0.04 ± 0.04 ± 0.04 ± 0.04 ± 0.06 ± 0.06b
ALP 5–131 107–215 112.80 117.70 85.10 96.50 82.20 90.40 73.20 67.10
(U/L) ± 10.15 ± 10.15 ± 7.24b± 7.24b± 7.56b± 7.56b± 5.73b± 5.73b
CK (U/L) 59–895 195–708 234.40 242.50 190.10 258.70 199.20 374.10 213.80 312.40
± 21.68 ± 21.68 ± 28.46b± 28.46 ± 61.45 ± 61.45b± 62.42 ± 62.42
TGs 29–291 35–114 33.00 32.70 39.30 36.30 41.30 40.90 36.20 38.80
(mg/dL) ± 3.66 ± 3.66 ± 3.11b± 3.11 ± 4.49b± 4.49b± 3.39 ± 3.39b
Table continues on next page

UA — 0.2–0.3 0.12 0.14 0.12 0.10 0.17 0.20 0.15 0.12
(mg/dL) ± 0.02 ± 0.02 ± 0.01 ± 0.01 ± 0.03 ± 0.03 ± 0.02 ± 0.02
Cr 0.5–1.6 0.7–1.3 0.79 0.99 0.81 0.83 1.00 1.06 0.97 1.00
(mg/dL) ± 0.08 ± 0.08 ± 0.02 ± 0.02 ± 0.04b± 0.04 ± 0.04b± 0.04
Gluc 70–138 32–134 108.40 108.20 106.60 108.30 101.70 98.30 97.30 97.90
(mg/dL) ± 2.15 ± 2.15 ± 2.15 ± 2.15 ± 2.62b± 2.62b± 1.48b± 1.48b
BUN — 8–111 16.11 19.94 15.23 18.22 19.87 21.47 17.98 19.19
(mg/dL) ± 1.59 ± 1.59 ± 0.92c± 0.92c± 1.07b± 1.07 ± 0.68 ± 0.68
TP (g/dL) 5.0–7.4 4.4–5.5 5.20 5.20 5.37 5.36 5.74 5.77 5.35 5.34
± 0.07 ± 0.07 ± 0.06b± 0.06b± 0.09b± 0.09b± 0.07 ± 0.07
ALB 2.7–4.4 3.0–3.8 3.15 3.18 3.42 3.45 3.50 3.54 3.37 3.38
(g/dL) ± 0.05 ± 0.05 ± 0.05b± 0.05b± 0.06b± 0.06b± 0.04b± 0.04b
Ca 8.9–11.4 9.4–12.1 11.37 11.46 10.82 10.86 11.46 11.51 11.04 10.99
(mg/dL) ± 0.08 ± 0.08 ± 0.06b± 0.06b0.09 ± 0.09 ± 0.11b± 0.11b
P (mg/dL) 2.5–6.0 7.9–10.6 6.31 6.57 5.70 5.55 5.83 5.99 4.69 4.73
± 0.18 ± 0.18 ± 0.15b± 0.15b± 0.25b± 0.25b± 0.25b± 0.25b
Glob 1.6–3.6 1.1–2.1 2.05 2.02 1.95 1.91 2.24 2.23 1.77 1.96
(g/dL) ± 0.07 ± 0.07 ± 0.05 ± 0.05 ± 0.05b± 0.05b± 0.15 ± 0.15
A/G 0.8–2.0 1.48–3.0 1.56 1.58 1.76 1.82 1.57 1.57 1.53 1.71
ratio ± 0.07 ± 0.07 ± 0.06b± 0.06b± 0.04 ± 0.04 ± 0.12 ± 0.12
B/C 4–27 10–123.33 20.38 20.36 18.83 22.28 19.93 20.29 18.61 19.31
ratio ± 0.69 ± 0.69 ± 1.34 ± 1.34 ± 0.68 ± 0.68 ± 0.69b± 0.69
aData shown are least squares mean ± SE.
bStatistically significant (P<.05) change from baseline within the supplement group based on a repeated measure, mixed model method with time point and supplement level as the fixed
factors.
cStatistically significant (P<.05) difference between the supplement groups based on a repeated measure, mixed model method with time point and supplement level as the fixed factors.
TABLE 2. Serum Chemistry Profile Parameters of Dogs Randomly Assigned to Either a Low or a High Concentration of Probiotic
Bifidobacterium animalis AHC7a(cont.)
Iams 18-
ANTECH Week-Old
Adult Puppy
Reference Reference Baseline (Week 0) Week 6 Week 12 Week 16
Range Range Low High Low High Low High Low High
Parameter 2009 2006 Concentration Concentration Concentration Concentration Concentration Concentration Concentration Concentration

therefore, it was not always possible to score
stool samples for individual dogs. If a dog was
observed defecating, its score was recorded; oth-
erwise, a single score was recorded for the co-
habitating dogs. Primary fecal scores were also
converted to a binary rating system by denoting
all scores >2 as “acceptable” (coded as 1) and all
scores ≤2 as “unacceptable” (coded as 0).
The dogs were monitored weekly for body
weight and every 3 weeks for BCS. In addition,
they received medical examinations (including
a complete physical examination, hematology,
and serum chemistry profile) at weeks 0, 6, 12,
and 16 to evaluate general health. Hematology
and serum chemistry profiles were evaluated in
relation to reference ranges provided by
ANTECH Diagnostics (Los Angeles, CA) for
adult dogs and those established by the PHNC
Clinical Laboratory (Lewisburg, OH) for rap-
idly growing puppies (Iams 18-week-old pup-
py reference range).
Fecal samples were collected in sterile con-
tainers at weeks 0, 6, 12, and 16 and submit-
ted to the PHNC Microbiology Laboratory
Veterinary Therapeutics • Vol. 11, No. 3, Fall 2010
E8
Figure 4. Changes in selected hematology parameters from baseline. All data are represented as least squares mean
(± SE) over time. Dotted lines represent ANTECH Diagnostics adult dog reference ranges (2009). * = Statistically
significant ( P<.05) change from baseline within the supplement group based on a repeated measure, mixed model
method with time point and supplement level as the fixed factors.
Low-level
High-level
Low-level
High-level
Low-level
High-level
Low-level
High-level
9.5
8.5
7.5
6.5
5.5
4.5
20
16
12
8
4
0
RBC (106/µL)
WBC (1000/µL)
21
19
17
15
13
11
60
55
50
45
40
35
30
Hgb (g/dL)
HCT (%)
Week
0612 16
Week
0612 16
Week
0612 16
Week
0612 16
RBC
Hemoglobin
WBC
Hematocrit
Changes From Baseline Hematology Parameters Were All Within ANTECH Adult Dog Reference Ranges

E9
(Lewisburg, OH) for enumeration of fecal bac-
teria. B. animalis AHC7 populations were enu-
merated using reverse-transcription poly-
merase chain reaction10,11 and the following
primers: universal, 5’ 6-FAM-CGTATTACC
GCGGCTGCTGGCAC-3’-TAMRA; for-
ward, 5’-TCCTACGGGAGGCAGCAGT-3’;
and reverse, 5’-GGACTACCAGGGTATC
TAATCCTGTT-3’ (all Applied Biosystems,
Inc., Foster City, CA); B. animalis AHC7, 5’
6FAM-CGGGTGGTGTCCCTTGCTG
GCT-3’-MGBNFQ; forward, 5’-GCTTC
CTTTCCTGGCCGT-3’; and reverse, 5’-
ACACCACAAGGGCGCAGG-3’ (all devel-
oped internally by Procter & Gamble Pet Care
based on 16s ribosomal sequence and pro-
duced by Applied Biosystems, Inc.).
Changes from baseline (week 0) within sup-
plement group and differences between sup-
plement groups were analyzed using a repeated
measure, mixed model method with time
point and supplement level as the fixed factors.
The analyses were conducted using PC SAS
9.2 (v8.2 SAS Institute, Inc, Cary, NC). All
data are reported as mean or least squares mean
± standard error. A Pvalue <.05 was declared
significant.
Body weight and feed intake were analyzed
with general linear models (GLM) procedures
of SAS to determine differences between treat-
ments. The model accounted for study week
and treatment group. Analysis of the microbial
population data was performed with GLM
procedures of SAS using repeated measures to
determine population changes over time. The
model accounted for sample timing as well as
individual animal within a treatment group.
All personnel involved in collection and analy-
sis of data were blind to treatment grouping.
■RESULTS
All dogs enrolled (11 males and 9 females)
completed all aspects of the study.
Baseline (Week 0)
No clinically or statistically significant differ-
ences were observed between the supplement
groups at baseline (week 0) for the following
parameters: mean BCS (3.0 for both groups;
data not shown), mean body weight (8.24 and
8.08 kg for the low- and high-concentration
groups, respectively; Figure 1), mean fecal
scores (3.87 and 3.94 for the low- and high-
concentration groups, respectively; data not
shown), mean weekly food intake (1.60 and
1.57 kg/wk/dog for the low- and high-concen-
tration groups, respectively; Figure 2), and
mean fecal bacteria population (Figure 3).
No clinically significant differences were ob-
served between the supplement groups at base-
line (week 0) for mean hematology and serum
chemistry profile parameters (Tables 1 and 2).
A statistically significant difference between the
supplement groups was observed at baseline for
MCHC (32.45 and 32.73 g/dL for the low-
and high-concentration groups, respectively [P
<.05]), but the mean baseline MCHC values
were within the adult and 18-week-old puppy
reference ranges. In addition, no clinically sig-
nificant abnormalities were seen during physi-
cal examinations in either group at baseline.
Supplementation and Observation Periods
Physical Examinations
Over the course of the study, no clinically
significant abnormalities were observed during
physical examinations. No clinically or statisti-
cally significant changes in heart rate, body
temperature, or respiration rate (data not
shown) were seen in either group over the
course of the study.
Hematology and Serum Chemistry Profiles
Complete Blood Count With Differential
Leukocyte Cell Count
No clinically significant abnormalities were
observed in hematology results over the course
R. L. Kelley, J. Soon Park, L. O’Mahony, D. Minikhiem, and A. Fix
(text continues on page E12)

Veterinary Therapeutics • Vol. 11, No. 3, Fall 2010
E10
Figure 5. Changes in selected serum chemistry profile parameters from baseline. All data are represented as least
squares mean (± SE) over time. Dotted lines represent ANTECH Diagnostics adult dog reference ranges (2009).
* = Statistically significant ( P<.05) change from baseline within the supplement group based on a repeated meas-
ure, mixed model method with time point and supplement level as the fixed factors. # = Statistically significant
(P<.05) difference between the supplement groups based on a repeated measure, mixed model method with time
point and supplement level as the fixed factors. (Figure continues on next page.)
Low-level
High-level
Low-level
High-level
Low-level
High-level
Low-level
High-level
Low-level
High-level
Low-level
High-level
105
85
65
45
25
5
1.7
1.5
1.3
1.1
0.9
0.7
0.5
BUN (mg/dL)
Creatinine (mg/dL)
140
130
120
110
100
90
80
70
60
150
125
100
75
50
25
0
Glucose (mg/dL)
ALKP (U/L)
Week
0612 16
Week
0612 16
Week
0612 16
Week
0612 16
Blood urea nitrogen
Glucose
Creatinine
Alkaline phosphatase
120
100
80
60
40
20
0
550
450
350
250
150
50
ALT (U/L)
CK (U/L)
Week
0612 16
Week
0612 16
Alanine aminotransferase Creatine kinase
Changes From Baseline Serum Chemistry Profile Parameters Were
All Within ANTECH Adult Dog Reference Ranges, Except Sodium at Week 12
#

E11
R. L. Kelley, J. Soon Park, L. O’Mahony, D. Minikhiem, and A. Fix
Figure 5. (cont.)
Low-level
High-level
Low-level
High-level
Low-level
High-level
Low-level
High-level
Low-level
High-level
Low-level
High-level
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
T_Protein (g/dL)
Albumin (g/dL)
3.4
3.0
2.6
2.2
1.8
1.4
1.0
160
155
150
145
140
135
Globulin (g/dL)
Sodium (mmol/L)
Week
0612 16
Week
0612 16
Week
0612 16
Week
0612 16
Total protein
Globulin
Albumin
Sodium
120
116
112
108
104
100
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
Chloride (mg/dL)
Phosphorus (mg/dL)
Week
0612 16
Week
0612 16
Chloride Phosphorus
Changes From Baseline Serum Chemistry Profile Parameters Were
All Within ANTECH Adult Dog Reference Ranges, Except Sodium at Week 12
#

of the study, although some changes from
baseline were detected statistically (Table 1;
Figure 4). Statistically significant mean
changes from baseline within each supplement
group were observed over the course of the
study for red blood cell count and hemoglo-
bin; these values were within the adult refer-
ence ranges but above the 18-week-old puppy
reference ranges. In addition, a statistically sig-
nificant mean change from baseline was found
at week 16 in hematocrit for the high-concen-
tration group; this value was within the adult
reference range but slightly above the 18-week-
old puppy reference range. All other statistical-
ly significant mean changes from baseline
within supplement group or mean differences
between supplement groups for hematology
parameters were within both reference ranges.
Serum Chemistry Profile
No clinically significant abnormalities were
observed in serum chemistry results over the
course of the study, although some changes
from baseline were detected statistically (Table
2; Figure 5). A statistically significant mean
change from baseline was observed within each
supplement group at week 12 for sodium that
was above both adult and puppy reference
ranges. Statistically significant mean changes
from baseline within each supplement group
were observed over the course of the study for
alkaline phosphatase and phosphorus; these
changes were within the adult reference ranges
but below the 18-week-old puppy reference
ranges. Statistically significant mean changes
from baseline within the supplement groups
were observed at some time points for chlo-
ride, creatine kinase, and total protein; these
changes were also within the adult reference
ranges but outside of the 18-week-old puppy
reference ranges. All other statistically signifi-
cant mean changes from baseline within sup-
plement group or mean differences between
supplement groups for serum chemistry profile
parameters were within both reference ranges.
Body Condition Scores
The mean BCS was 3.0 in both supplement
groups at baseline and remained near this ide-
al score in both groups for the duration of the
study. Mean scores ranged from 3.10 to 3.30
in the low-concentration group and 3.30 to
3.47 in the high-concentration group during
the supplementation and observation periods.
Body Weight and Food Intake
Body weight increased gradually over the
course of the study in both supplement groups,
as expected (Figure 1). Weekly food intake was
similar in both groups (Figure 2). No statisti-
cally significant differences were noted be-
tween the groups at any time point for either
of these parameters.
Fecal Scores
Mean fecal scores were maintained at approx-
imately 4 (firm, well-formed stool) throughout
the study in both groups, although statistically
significant differences between the low- and
high-concentration groups were seen at weeks 2
(3.85 and 3.99, respectively [P<.01]), 3 (3.79
and 3.97, respectively [P<.0001]), 4 (3.81 and
3.95, respectively [P<.01]), and 9 (3.85 and
3.97, respectively [P<.05]) of the supplementa-
tion period and at week 15 (3.85 and 3.95, re-
spectively [P<.05]) of the observation period.
All primary fecal scores converted to a binary
rating system (acceptable versus unacceptable)
were denoted “acceptable” for both probiotic
supplement groups.
Fecal Bacteria Populations
As expected, supplementation with B. ani-
malis AHC7 resulted in statistically significant
(P≤.05) increases in the overall percentage of
B. animalis AHC7 present in fecal samples in
Veterinary Therapeutics • Vol. 11, No. 3, Fall 2010
E12

E13
the high-concentration group during the sup-
plementation period (weeks 6 and 12), with
percentages returning to baseline during the
observation period (Figure 3). In the low-con-
centration group, B. animalis AHC7 increased
numerically during the supplementation peri-
od but had no statistical difference from base-
line levels at 6 weeks (P<.11) and 12 weeks (P
<.13), with levels at the end of the observation
period (16 weeks) being similar to baseline
levels.
Total bacterial concentrations were not af-
fected by either low- or high-concentration
supplementation with B. animalis AHC7. The
percentage of total Lactobacillus was not affect-
ed by B. animalis AHC7 supplementation, al-
though overall levels were slightly reduced nu-
merically at weeks 12 and 16 (data not shown).
■DISCUSSION
Commercially available probiotics for pets
are becoming more common, even though lit-
tle information exists regarding their safety. Al-
though probiotic supplementation is generally
considered harmless, some safety concerns
have been documented in humans.7The pres-
ent study concentrated primarily on evaluating
the safety and tolerance of dietary supplemen-
tation with the probiotic B. animalis AHC7
fed to growing dogs, a group most likely to ex-
hibit problems if any were to occur, at a high-
er dose and longer duration than tested in pre-
vious studies.8,9
No clinically significant abnormalities were
observed during physical examinations of the
dogs fed up to 5 ×1010 CFU/day for at least 12
consecutive weeks. Although statistically sig-
nificant changes from baseline that were out-
side the reference ranges for some hematology
and clinical chemistry parameters were seen in
both supplement groups, none of these
changes were considered to be clinically signif-
icant. Hematology and clinical chemistry val-
ues are known to change as healthy puppies
mature, and puppy values may lie outside the
established normal ranges for adults. In pup-
pies younger than 8 months, serum alkaline
phosphatase of bone origin is commonly up to
twice the adult level,12and packed cell volume
increases to reach a normal, mature level be-
tween 2 and 6 months of age.13 The statistical-
ly significant differences from baseline ob-
served in this study generally followed
expected age-related trends.
The serum sodium concentration was in-
creased in both supplement groups at 6 and 12
weeks, but it did not reach clinically significant
hypernatremia (>160 mEq/L for dogs).14 Base-
line serum sodium levels in the dogs in this
study were at the higher end of the reference
range, so the increases were relatively modest.
Although neither this nor other studies have
revealed a cause-and-effect association of pro-
biotic supplementation and increased serum
sodium concentration, probiotics have been
reported to enhance absorption of other
minerals.15,16
Mean BCS was maintained at approximate-
ly 3.0 (ideal body condition) throughout the
study in both supplement groups. As expected,
these young dogs continued to grow during
the study period as evidenced by increasing
body weight over time. The growth rate ob-
served in this study was similar to that seen
with growing beagles of similar age.17 Statisti-
cally significant differences between the low-
and high-concentration groups were observed
at some time points for mean fecal scores.
However, it is unlikely that fractional differ-
ences of the magnitude observed for the group
means would be detected for individual sub-
jects. Mean fecal scores were maintained at
approximately 4 (firm, well-formed stool)
throughout the study in both groups, and not-
ed statistical differences were not considered to
be clinically significant.
R. L. Kelley, J. Soon Park, L. O’Mahony, D. Minikhiem, and A. Fix

As expected, supplementation with B. ani-
malis AHC7 resulted in statistically significant
(P≤.05) increases in the overall percentage of
B. animalis AHC7 present in fecal samples
during the 12-week supplementation period in
the high-concentration group, as well as an in-
creasing trend in the low-concentration group.
Having a larger number of dogs might have
overcome the high variability in fecal bacterial
population at the low concentration of supple-
mentation to enable detection of significant in-
creases in both groups during supplementa-
tion. As expected, percentages returned to
baseline during the 4-week observation period
in both groups.
■CONCLUSION
This study demonstrated that oral adminis-
tration of canine-derived B. animalis AHC7
once per day at a dose of up to 5 ×1010 CFU
for at least 12 consecutive weeks was well toler-
ated by growing dogs with no safety concerns.
■REFERENCES
1. Schrezenmeir J, de Vrese M. Probiotics, prebiotics,
and synbiotics—approaching a definition. Am J Clin
Nutr 2001;73(suppl):361S-364S.
2. Weese JS. Evaluation of deficiencies in labeling of
commercial probiotics. Can Vet J 2003;44:982-983.
3. Boyle RJ, Robins-Browne RM, Tang MLK. Probiot-
ic use in clinical practice: what are the risks? Am J Clin
Nutr 2006;83:1256-1264.
4. Hammerman C, Bin-Nun A, Kaplan M. Safety of
probiotics: comparison of two popular strains. BMJ
2006;333:1006-1008.
5. Ishibashi N, Yamazaki S. Probiotics and safety. Am J
Clin Nutr 2001;73(suppl):465S-470S.
6. Vlieger AM, Robroch A, van Buuren S, et al. Toler-
ance and safety of Lactobacillus paracasei ssp. paracasei
in combination with Bifidobacterium animalis ssp. lac-
tis in a prebiotic-containing infant formula: a ran-
domized controlled trial. Br J Nutr 2009;102:869-
875.
7. Vogel G. Deaths prompt a review of experimental
probiotic therapy. Science 2008;319:557.
8. O’Mahony D, Murphy KB, MacSharry J, et al. Por-
trait of a canine probiotic Bifidobacterium—from gut
to gut. Vet Microbiol 2009;139:106-112.
9. Kelley RL, Minikhiem D, Kiely B, et al. Clinical ben-
efits of probiotic canine-derived Bifidobacterium ani-
malis strain AHC7 in dogs with acute idiopathic diar-
rhea. Vet Ther 2009;10:121-130.
10. Sakata S, Kitahara M, Sakamoto M, et al. Unification
of Bifidobacterium infantis and Bifidobacterium suis as
Bifidobacterium longum.Int J Syst Evol Microbiol
2002;52:1945-1951.
11. Mayer HK, Amtmann E, Philippi E, et al. Molecular
discrimination of new isolates of Bifidobacterium ani-
malis subsp. lactis from reference strains and commer-
cial probiotic strains. Int Dairy J 2007;17:565-573.
12. Willard MD, Twedt DC. Gastrointestinal, pancreat-
ic, and hepatic disorders. In: Willard MD, Tvedten
H, eds. Small Animal Clinical Diagnosis by Laboratory
Methods. 4th ed. St. Louis: Saunders; 2004:208-246.
13. The hematopoietic and lymphoid systems. In:
Hoskins JD. Veterinary Pediatrics: Dogs and Cats from
Birth to Six Months. 3rd ed. Philadelphia: WB Saun-
ders; 2001:300-343.
14. DiBartola SP, Green RA, de Morais HSA, Willard
MD. Electrolyte and acid-base disorders. In: Willard
MD, Tvedten H, Turnwald GH, eds. Small Animal
Clinical Diagnosis by Laboratory Methods. 3rd ed.
Philadelphia: Saunders; 1999:93-108.
15. Gilman J, Cashman KD. The effect of probiotic bac-
teria on transepithelial calcium transport and calcium
uptake in human intestinal-like Caco-2 cells. Curr Is-
sues Intest Microbiol 2006;7(1):1-5.
16. Borthakur A, Gill RK, Tyagi S, et al. The probiotic
Lactobacillus acidophilus stimulates chloride/hydroxyl
exchange activity in human intestinal epithelial cells. J
Nutr 2008;138(7):1355-1359.
17. Procter & Gamble Pet Care, Lewisburg, OH. Un-
published data.
Veterinary Therapeutics • Vol. 11, No. 3, Fall 2010
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