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Effects of Lactobacillus acidophilus DSM13241
as a probiotic in healthy adult cats
Zoe V. Marshall-Jones, PhD; Marie-Louise A. Baillon, PhD; Julie M. Croft, BSc; Richard F. Butterwick, PhD
Objective⎯To evaluate the effect of dietary supple-
mentation with the probiotic strain Lactobacillus aci-
dophilus DSM13241 in healthy adult cats.
Animals⎯15 adult cats.
Procedures⎯Cats were fed a nutritionally complete
dry food for 5 weeks. Fecal character was assessed
daily, and a single fecal sample and 3-mL blood sam-
ple were collected for bacterial enumeration and
hematologic analysis, respectively. Cats were then
fed the same diet supplemented with L acidophilus
DSM13241 (2 X 108 CFU/d) for 4.5 weeks. Repeat
fecal and hematologic measurements were taken
prior to the return to control diet for a 4-week period.
Results⎯The probiotic species was recovered from
feces, demonstrating survival through the feline gas-
trointestinal tract. Probiotic supplementation was
associated with increased numbers of beneficial
Lactobacillus and L acidophilus groups in feces and
decreased numbers of Clostridium spp and
Enterococcus faecalis, indicating an altered bacterial
balance in the gastrointestinal tract microflora. Fecal
pH was also decreased suggesting a colonic environ-
ment selective for the beneficial lactic acid bacterial
population. Systemic and immunomodulatory effects
were associated with administration of L acidophilus
DSM13241 including altered cell numbers within
WBC subsets and enhanced phagocytic capacity in
the peripheral granulocyte population. In addition,
plasma endotoxin concentrations were decreased
during probiotic feeding, and RBCs had a decreased
susceptibility to osmotic pressure.
Conclusions and Clinical Relevance⎯Probiotic strain
L acidophilus DSM13241 fed at 2 X 108 CFU/d can alter
the balance of gastrointestinal microflora in healthy
cats. Furthermore, administration of this probiotic
results in beneficial systemic and immunomodulatory
effects in cats. (Am J Vet Res 2006;67:1005–1012)
C ommensal gastrointestinal tract microflora are vital
to gastrointestinal health, aiding the host in diges-
tion, nutrient metabolism, and vitamin production and
in restricting colonization by pathogenic bacteria.1,2
Microbial populations in the gastrointestinal tract, how-
ever, are susceptible to change induced by factors such
as poor nutrition, stress, gastrointestinal infections,
antibiotic administration, predisposing illness, and
immunosuppression. These fluctuations may impact the
health of the host; therefore, enhancement of the colonic
microflora by dietary interventions such as probiotics
may be used to maintain gastrointestinal health.
Received August 24, 2005.
Accepted November 22, 2005.
From the Waltham Centre for Pet Nutrition, Waltham-on-the-
Wolds, Melton Mowbray, Leicestershire, LE14 4RT, UK.
Address correspondence to Dr. Marshall-Jones.
AJVR, Vol 67, No. 6, June 2006
ABBREVIATIONS
FISH Fluorescence in situ hybridization
MRS De Man, Rogosa, and Sharpe
rRNA Ribosomal RNA
Probiotics are defined as live microbial feed sup-
plements, which beneficially affect the host animal by
improving its gastrointestinal microbial balance.3 The
bacterial species most often used as health-promoting
probiotics are Lactobacillus and Bifidobacterium spp,
although probiotic yeasts and Enterococcus spp are also
used to promote weight gain in production animals.3-5
Probiotics are increasingly used therapeutically for
the treatment of gastrointestinal complaints, including
antimicrobial and stress-associated diarrhea, as well as
bacterial and viral diarrheal diseases.5-7 Dietary supple-
mentation with probiotics has also been reported to
alleviate inflammatory bowel disease and reduce the
occurrence of colonic carcinomas.8,9
These microorganisms may exert positive effects
on the host by various means. Firstly, they increase
numbers of beneficial bacteria such as lactobacilli and
bifidobacteria, compared with putrefying or potential-
ly detrimental bacteria.10,11 This improved microbial
balance may occur through inhibition of detrimental
species by competition for nutrients and gastrointesti-
nal binding sites. However, the production of metabo-
lites such as lactic acid, which reduce the pH of the
colon favoring growth of other lactic acid bacteria, and
secretion of antimicrobial peptides that directly target
bacterial pathogens may also be involved.12-14 Secondly,
probiotics may regulate host immune function.
Although the exact mechanisms underlying immune
effects are currently unclear, lactobacilli and bifidobac-
teria are known to modulate the expression of
immunoregulatory cytokines by dendritic cells and
macrophages.15-17
Probiotics are commonly used in production ani-
mals, and more recently, interest in their usage for pro-
motion of companion animal health has increased.
Although several studies have investigated probiotic
usage in dogs, few have shown beneficial effects.18,19
Administration of Lactobacillus acidophilus DSM13241
has been shown to improve the gastrointestinal micro-
bial balance and induce immunostimulatory effects in
adult dogs,20 whereas dietary supplementation with
another L acidophilus strain was observed to stimulate
appetite and growth in puppies up to 19 weeks of age.21
The probiotic Enterococcus faecium (SF68/NCIMB
10415) was reported to stimulate immune function in
young dogs,22 with a second study18 on the same E fae-
cium probiotic reporting modification of the canine
fecal microflora. In that study,18 however, the beneficial
1005
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effect of the probiotic remained questionable with fecal
Salmonella and Campylobacter spp higher in most dogs
following probiotic supplementation.
Studies on probiotic usage in cats have not been
reported to date to our knowledge, and because of dif-
ferences in host physiologic characteristics and diet,
probiotic efficacy in cats cannot be extrapolated from
studies in dogs. In addition, differences exist in the res-
ident colonic microflora, which, in cats, is thought to
comprise high numbers of anaerobic bacteria similar to
those representing bacterial overgrowth in the small
intestine of humans and dogs.23
The purpose of the study reported here was to
determine whether dietary supplementation with the
probiotic L acidophilus DSM13241a would induce
health-related effects in adult cats. The probiotic was
incorporated into dry cat food at a concentration deliv-
ering > 108 CFU/d.
Materials and Methods
Study design⎯Fifteen domestic shorthair cats were
involved in a 3-phase longitudinal study to assess the effects
of supplementation with the probiotic strain L acidophilus
DSM13241.a A within-subject study design was used to
remove an important source of between-subject variation,
with each cat representing its own control. Cats were fed a
nutritionally complete commercial dry diet for a period of 5
weeks (baseline phase) before switching to the same diet
supplemented with the probiotic strain for 4.5 weeks (probi-
otic phase). Following probiotic supplementation, cats
returned to the base diet without probiotics for a period of 4
weeks (postprobiotic phase). Fecal samples were collected in
the final 2 weeks of each phase for the assessment of gas-
trointestinal health status. Small-volume blood samples were
collected 5 days prior to the end of each study phase for
assessment of systemic effects and immune status.
Comparisons between the data collected during each phase
allowed assessment of the effects of supplementation with
L acidophilus DSM13241 in cats.
Kibble preparations, packaging, and validation of probi-
otic inclusion was conducted as described by Baillon et al.20
The probiotic strain was incorporated into the supplemented
diet to an inclusion concentration analyzed at 4.1 X 109
CFU/kg of foodstuff at the start of the study, decreasing to 3.2
X 109 CFU/kg by the end of the study.
Animals and husbandry considerations⎯Cats
involved in the study were between 4 and 5.5 years old
(mean ± SD, 4.5 ± 0.4 years), weighing between 3.6 and 7.3
kg (3.6 ± 1.1 kg). For ease of feeding and feces measure-
ments, cats were housed individually in 2-room, environ-
mentally enriched lodges as described by Loveridge.24 Full
animal welfare considerations were in place. The study was
approved by the Waltham Centre for Pet Nutrition Ethical
Review Committee, in accordance with the UK Home Office
Animals (Scientific Procedures) Act of 1986. Cats were
socialized daily, and fresh water was available at all times.
Cats were fed once daily at energy levels required to main-
tain body weight (food offered, 58.73 ± 10.24 g). Probiotic
administration equated to a daily intake of between 1.2 X 108
CFU and 2.8 X 108 CFU dependent on the individual
requirements. Feeding occurred over a 2-hour period to
minimize exposure of the probiotic to air and humidity.
Food offered and refused was recorded for each cat daily, and
body weight was measured weekly. Prior to the start of the
study and during each study phase, cats received a physical
examination and health assessment. All cats were healthy
and had no record of chronic illness.
1006
Samples and measurements⎯Fecal quality was
assessed daily by use of a feces scoring systemb as described
by Rolfe et al.25 Trained assessors graded feces on a scale of 1
to 5, in which grade 1 represented dry crumbly feces and
grade 5 represented diarrhea. Each major sector (1 through
5) was subdivided into 4 subsectors to allow accurate scoring
of fecal form. Mean fecal score was calculated for each cat
during each phase of the study. During the final 2 weeks of
each phase, fecal samples were collected within 30 minutes
of production for assessment of bacterial populations, pH,
and ammonia and hydrogen sulfide contents.
Fecal bacterial populations were enumerated by use of
selective bacterial culture and FISH. For selective culture,
fecal material was prepared by 10% (wt/vol) dilution in 50%
(vol/vol) maximum recovery diluent.c Anaerobic culture of
total anaerobes, lactobacilli, and clostridia was supported by
the addition of 0.025% (wt/vol) cysteine hydrochloride and
prior reduction of reagents in anaerobic conditions.
Duplicate serial dilutions of the fecal solution were prepared
to a dilution of 10–8, and 50-µL aliquots were spread plated
onto the appropriate media.c Total anaerobes were selected
on fastidious anaerobe agar; lactobacilli were selected on
MRS agar acidified to pH 5, and clostridia were selected by
use of a Clostridium perfringens agar base containing tryp-
tose-sulfite-cycloserine selective supplement. Inoculated agar
plates were incubated at 38°C under anaerobic conditions
(10% H2, 10% CO2, and 80% N2) for 18 to 48 hours depend-
ing on bacterial growth characteristics. Aerobic culture of
enterococci and coliforms was supported by selection on
K-F Streptococcus agar and MacConkey No. 3 agar, respec-
tively; these cultures were incubated under aerobic condi-
tions at 38°C for 18 to 48 hours.
Putative lactobacilli selected on acidified MRS medium
were retained for characterization by biochemical profiling,d
through which taxonomic classification is determined by the
characteristic ability of an isolate to ferment 49 carbohydrate
substrates. Isolates that had fermentation patterns character-
istic of the probiotic strain were characterized by DNA fin-
gerprinting of the 16S rRNA gene.e An automated ribotyping
systemf was used in which genomic DNA was extracted and
digested with restriction endonucleases; the resulting frag-
ments were separated by gel electrophoresis. Hybridization
with fluorescein-labeled probes designed against the 16S
rRNA gene allowed observation of the DNA banding pattern,
which was analyzed by use of proprietary software.f
Because growing evidence suggests that selective culture
for assessment of microbial ecology is inadequate,26 measure-
ment of bacterial populations by the molecular FISH method
was used to further enumerate fecal bacteria. A series of 5
cyanine-3 labeled DNA probesg targeted against the 16S or
23S rRNA were used in the analysis of Lactobacillus spp,
L acidophilus, bifidobacteria, clostridia, and Enterococcus fae-
calis (Appendix).27-30
Briefly, 3 g of feces was diluted 10% (wt/vol) in PBS solu-
tion, and particulate material was removed by centrifugation
at 1,000 X g for 2 minutes. The supernatant was diluted 1:4
in 4% paraformaldehyde. Bacterial cells were harvested from
the supernatant by centrifugation at 12,500 X g for 5 min-
utes. The harvested cells were washed in PBS solution and
suspended in a 900-µL volume of 50% (vol/vol) ethanol-PBS
solution. Cellular material was then hybridized overnight at
45oC with 1 µg of a cyanine-3–labeled DNA probe and with
the total bacterial stain, 4'6-diamidino-2-phenylindole.
Fluorescent-labeled and stained cells were counted by use of
fluorescence microscopy.h
Fecal pH was measured in triplicate in fecal water
extracted by centrifugation of 10 g of fresh feces at 40,000 X
g for 2 hours. Fecal ammonia was measured in feces that was
diluted 5% (wt/vol) in sterile distilled water containing 20%
AJVR, Vol 67, No. 6, June 2006
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(vol/vol) ammonia pH adjusting solutioni by use of an
ammonia electrode.i Total sulfide (H2S and HS–) production
in feces was measured by use of the methylene blue method.
Briefly, 0.3 g of feces was diluted 1:10 in prereduced sodium
phosphate buffer (pH, 7.4) and agitated to mix. The resulting
slurry was used as an inoculum diluted 10% (vol/vol) in
anaerobe basal brothc, and the culture was incubated at 38°C
under anaerobic conditions for 24 hours. Cultures were
diluted 1:4 in sterile distilled water, and particulate material
was removed by centrifugation at 13,800 X g for 15 minutes.
Total sulfides were detected by use of the hydrogen sulfide
reagent setj and colorimetric detection by spectrophotometry
at a wavelength of 670 nm. Sulfides were measured in parts
per million against an Na2S standard curve.
A 3-mL blood sample was collected by venipuncture of
the cephalic vein during the final week of each study phase.
Sample analyses included measurement of acellular immune
factors and hemogram,k which included a differential WBC
count. Serum biochemical analysis included serum concen-
trations of total protein, albumin, urea, creatinine, choles-
terol, calcium, and inorganic phosphorus; serum activities of
alanine aminotransferase, alkaline phosphatase, and aspar-
tate aminotransferase were also analyzed. Serum immuno-
globin concentrations (IgG, IgA, and IgM) were determined
via commercially available radial immunodiffusion assays,l
and concentrations of the serum acute-phase reactants hap-
toglobin and C-reactive protein were measured via commer-
cially available immunoassays.m Total serum nitric oxide con-
centrations were determined by enzymatic conversion of
nitrate to nitrite followed by colorimetric detection of diazo-
tization products of the Griess reactionn by spectrophotome-
try at a wavelength of 550 nm.
Assessment of RBC susceptibility to osmotic pressure31
was performed on whole-blood samples collected in EDTA.
Blood samples (20 µL) were diluted to 1% (vol/vol) in sodi-
um chloride solution of various concentrations (0.55%,
0.6%, 0.65%, 0.7%, and 0.9% [wt/vol]), and samples were
incubated for 20 minutes at 22°C. Cellular material was
removed by centrifugation at 500 X g, and the optical densi-
ty of the supernatant was determined by spectrophotometry
at a wavelength of 540 nm. Exposure to 0.65% (wt/vol) sodi-
um chloride resulted in hemolysis of > 0% and < 100% for all
samples and was therefore used for data analysis purposes.
Endotoxin (bacterial lipopolysaccharide) concentra-
tions in serum from 1-mL whole-blood samples collected in
pyrogen-free vessels were determined at external laborato-
rieso by use of the Limulus amebocyte lysate test32 after
removal of endotoxin inhibitors by heating at 75°C for 30
minutes. Leukocyte phagocytic activity was investigated in
vitro by use of a commercially available assayp and flow
cytometry.q Heparinized whole blood was challenged with
opsonized fluorescein isothiocyanate–labeled Escherichia coli
and incubated at 37°C to support phagocytosis. Following
quenching of extracellular bacteria, RBCs were lysed and
fixed and WBC DNA was stained by use of propidium iodide
solution. Phagocytic activity of granulocyte and monocyte
Table 1⎯Mean ⫾ SD number of selected bacterial populations recovered from feces by bacterial cul-
ture during baseline, probiotic supplementation, and after probiotic treatment phases.
Bacterial numbers (Log10/g)
Bacterial population Baseline
Lactobacilli 6.83 ⫾ 0.68
Total anaerobes 10.04 ⫾ 0.86
Clostridia 9.14 ⫾ 0.97a
Coliforms 5.52 ⫾ 1.00a
Enterococci 2.70 ⫾ 1.44a
Values represent data from 15 cats.
a,b
Significant (P ⬍ 0.05) difference among phases.
AJVR, Vol 67, No. 6, June 2006
populations was measured by flow cytometry by use of the
blue-green excitation light (488 nm).r In addition to analysis
of phagocytic activity of the individual phagocyte popula-
tions, the median fluorescence intensity was measured for
comparison of mean number of bacteria phagocytosed per
WBC.
⎯Data from the 3 phases were ana-
Statistical analysis⎯
lyzed by use of a 2-factor general linear model to take into
account the observation of the same cat in each of the follow-
ing phases: baseline, probiotic treatment, and after probiotic
treatment. Where measurements were taken over 2 study
phases, data were analyzed by use of a paired Student t test.s
In the case of bacterial populations and species counts, data
were logarithmically transformed prior to analysis, and fecal
scores and body weights were averaged over each phase for
each cat prior to analysis. Treatment effects found to be sig-
nificant at the 5% significance level were followed up by use
of the Student-Newman-Keuls multiple range test. Data were
stored electronically and analyzed by use of the multifactor
ANOVA procedure with proprietary analysis software.t Results
are expressed as mean ± SD values.
Results
Health status⎯Probiotic supplementation was
not associated with changes in the health status of cats,
as demonstrated by findings on physical examination,
CBC determination, and serum biochemical analysis.
Food intake was consistent throughout the study
(baseline, 47.2 ± 11 g; probiotic, 47.6 ± 10.8 g; after
probiotic, 47.8 ± 9.2 g; P = 0.902), and body weight
was not significantly (P = 0.178) altered by probiotic
feeding (baseline, 5.27 ± 1.06 kg; probiotic, 5.22 ±
1.09 kg; after probiotic, 5.21 ± 1.12 kg).
Fecal measurements⎯Fecal quality was excellent,
and fecal scores remained unchanged throughout the
study (baseline, 2.03 ± 0.14; probiotic, 2.02 ± 0.16;
after probiotic 2.06 ± 0.12; P = 0.388). Hydrogen sul-
fide (baseline, 34.21 ± 10.04 µg/g; probiotic, 31.18 ±
10.86 µg/g; after probiotic, 36.57 ± 10.52 µg/g; P =
0.394) and ammonia (baseline, 11.48 ± 4.89mM; pro-
biotic 10.40 ± 6.03mM; after probiotic, 9.13 ±
4.32mM; P = 0.239) concentrations were also constant
among fecal samples throughout the study. Fecal pH
had a slight but significant (P = 0.017) decrease across
the course of the study (baseline, 6.73 ± 0.34; probiot-
ic, 6.57 ± 0.45; after probiotic, 6.48 ± 0.41).
Assessment of fecal bacterial populations by selec-
tive culture (Table 1) revealed that clostridia numbers
were significantly reduced during the probiotic-feeding
phase and increased following the cessation of probiot-
ic supplementation, whereas coliform and Enterococcus
populations decreased during the course of the study.
Probiotic After probiotic P value
6.78 ⫾ 0.61 6.03 ⫾ 1.10 0.171
9.86 ⫾ 0.61 9.73 ⫾ 0.58 0.531
8.34 ⫾ 0.83b 8.63 ⫾ 0.36a,b 0.043
4.83 ⫾ 1.94a 4.23 ⫾ 1.38b 0.018
2.06 ⫾ 1.61a 0.55 ⫾ 1.17b ⬍ 0.001
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No significant changes in numbers of culturable lacto- changes were observed in the lymphocyte population
bacilli and total anaerobes were observed by selective with a reduction in cell numbers across the course of
culture. the study and a similar increase in numbers of
Biochemical profiling was used to assess the car- eosinophils (Table 3). The proportion of the monocyte
bohydrate fermentation pattern of randomly selected and granulocyte populations having phagocytic activi-
isolates cultured on MRS agar from each cat. During ty in vitro remained unchanged, compared with base-
the baseline, phase isolates were predominantly identi- line numbers during probiotic feeding (Table 4).
fied as Lactobacillus plantarum, Lactobacillus brevis, and During the postprobiotic phase, however, the percent-
Pediococcus pentosaceus, while isolates with profiles age of cells from both populations that were active in
characteristic of the probiotic strain were not identi- phagocytosis was slightly, yet significantly, reduced,
fied. During the probiotic feeding phase, however, 11 compared with baseline. The mean fluorescent intensi-
of the 28 isolates tested had fermentation profiles char- ty, which correlates to the mean number of bacteria
acteristic of the probiotic strain. Molecular fingerprint- phagocytosed per WBC, was unchanged in the mono-
ing of the 16S rRNA gene revealed that these isolates cyte population throughout the study. However, mean
were identical to the probiotic strain (Figure 1). On numbers of bacteria ingested by the granulocyte popu-
returning to the base diet, biochemical profiling sug- lation increased significantly in response to probiotic
gested a return to Lactobacillus populations character- administration and again following the cessation of
istic of the baseline period. probiotic feeding. The acellular immune factors IgG,
Molecular methods for bacterial enumeration had IgM, and acute-phase reactants serum amyloid A, and
several differences in bacterial populations, compared haptoglobin remained constant throughout the study,
with bacterial culture. Numbers of lactobacilli as with serum nitric oxide concentrations also unchanged
observed by FISH increased significantly during probi- by administration of the probiotic supplement.
otic-feeding in actual numbers and as a percentage of The susceptibility of RBCs to hemolysis on expo-
the total bacterial population (Table 2). Similarly, num- sure to 0.65% (wt/vol) NaCl solution was reduced sig-
bers of the L acidophilus group (also including nificantly (P = 0.028) throughout the course of the
Lactobacillus crispatus, Lactobacillus gallinarum, and study (baseline, 17.14 ± 11.32%; probiotic, 14.18 ±
Lactobacillus helveticus) increased signifi-
cantly during probiotic feeding as measured
by FISH. Numbers of Bifidobacterium spp
decreased as a percentage of the total popu-
lation in the supplementation phase and
increased to baseline numbers in the post-
probiotic phase. As observed by bacterial
culture, FISH analysis revealed a reduction
in the opportunistic pathogen E faecalis, in
bacterial numbers and percentage popula-
tion, throughout the course of the study.
Technical difficulties were encountered in
assessment of fecal clostridia by FISH dur-
ing the baseline period. However, consistent
with observations by selective culture, after
probiotic administration, numbers of
clostridia were increased significantly, com-
pared with those during supplementation.
Blood parameters⎯Results of blood
biochemical analysis and CBC determina-
tion were within reference ranges and Figure 1⎯Molecular fingerprint of the 16S rRNA of the probiotic strain (A) and 4
revealed no significant changes associated fecal isolates (B through E) cultured on MRS agar during probiotic supplementa-
with probiotic supplementation. Significant tion. kb = Kilobase.

Table 2⎯Mean ⫾ SD number of fecal bacterial populations as determined by use of FISH expressed in actual numbers and as a per-
centage of total population during baseline, probiotic supplementation, and after probiotic treatment phases.

Absolute numbers (log10/g) Total population (%)

Bacterial spp Baseline Probiotic After probiotic P value Baseline Probiotic After probiotic P value
Lactobacillus spp 8.21 ⫾ 0.32a
8.57 ⫾ 0.14 b
8.19 ⫾ 0.20 a
⬍ 0.001 1.31 ⫾ 0.95a,b
1.93 ⫾ 1.00 b
1.03 ⫾ 0.49a
0.022
L acidophilus 0.47 ⫾ 1.68a 7.25 ⫾ 0.68b 0.40 ⫾ 1.57a ⬍ 0.001 0.0003 ⫾ 0.00a 0.2 ⫾ 0.23b 0.0005 ⫾ 0.00a ⬍ 0.001
Bifidobacterium spp 7.34 ⫾ 0.26 7.24 ⫾ 0.14 7.36 ⫾ 0.21 0.224 0.16 ⫾ 0.07a 0.09 ⫾ 0.04b 0.15 ⫾ 0.09a 0.036
Enterococcus faecalis 6.80 ⫾ 2.05a 4.10 ⫾ 3.69b 0.49 ⫾ 1.91b ⬍ 0.001 0.15 ⫾ 0.09a 0.05 ⫾ 0.06b 0.01 ⫾ 0.04b ⬍ 0.001
Clostridium spp ND 8.13 ⫾ 0.48a 8.76 ⫾ 0.34b 0.011 ND 1.27 ⫾ 2.06a 4.03 ⫾ 1.98b 0.003
ND = Not determined.
See Table 1 for remainder of key.

1008 AJVR, Vol 67, No. 6, June 2006

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Table 3⎯Mean ± SD WBC population numbers and acellular immune factor concentrations in blood
samples obtained during baseline, probiotic supplementation, and after probiotic treatment phases.

Immune factor Baseline Probiotic After probiotic P value

9
WBCs (X 10 /L) 11.50 ⫾ 2.27 11.21 ⫾ 2.99 11.05 ⫾ 3.63 0.828
Neutrophil (X 109/L) 5.94 ⫾ 1.67 5.96 ⫾ 1.67 5.91 ⫾ 2.19 0.926
Lymphocyte (X 109/L) 4.89 ⫾ 1.81a 4.36 ⫾ 1.94a 4.05 ⫾ 1.69b 0.043
Monocyte (X 109/L) 0.17 ⫾ 0.17 0.19 ⫾ 0.17 0.14 ⫾ 0.18 0.923
Eosinophil (X 109/L) 0.33 ⫾ 0.27a 0.70 ⫾ 0.37b 0.95 ⫾ 0.71b 0.002

IgG (mg/mL) 9.37 ⫾ 2.65 8.61 ⫾ 1.89 9.47 ⫾ 2.64 0.154

IgM (mg/mL) 1.05 ⫾ 0.40 0.96 ⫾ 0.35 0.96 ⫾ 0.35 0.200
Serum amyloid A (µg/mL) 1.48 ⫾ 0.06 1.56 ⫾ 0.05 1.54 ⫾ 0.06 0.508
Haptoglobin (mg/mL) 1.32 ⫾ 0.53 1.32 ⫾ 0.62 1.21 ⫾ 0.68 0.288
Nitric oxide (µM) 17.4 ⫾ 14.8 21.5 ⫾ 17.3 22.2 ⫾ 17.0 0.254
See Table 1 for key.

Table 4⎯Mean ± SD values of phagocytic activity of monocyte and granulocyte populations during
baseline, probiotic supplementation, and after probiotic treatment phases.

Phagocytic activity Baseline Probiotic After probiotic P value

Monocytes
Proportion of 91.94 ⫾ 2.80a 91.70 ⫾ 5.20a 79.13 ⫾ 6.91b ⬍ 0.001
population (%)

Fluorescence 557.42 ⫾ 129.56 546.05 ⫾ 150.86 606.56 ⫾ 82.19 0.273

intensity

Granulocytes
Proportion of 98.30 ⫾ 1.74a 95.77 ⫾ 5.84a 89.99 ⫾ 5.18b ⬍ 0.001
population (%)

Fluorescence 448.11 ⫾ 126.08a 581.79 ⫾ 128.38b 780.33 ⫾ 131.61b ⬍ 0.001

intensity
See Table 1 for key.

that may be considered beneficial to the

Figure 2⎯Mean plasma endotoxin concentrations in blood samples from 15
cats during the baseline (closed bar), probiotic feeding (hatched bar), and post- the study. This suggests a mechanism not
probiotic (open bar) phases. a,bSignificant (P < 0.001) difference between phases. requiring direct action of the probiotic or
10.60%; after probiotic, 11.83 ± 9.66%). Plasma endo-
toxin concentration was reduced during probiotic sup-
plementation with concentrations below the detection
limit in 6 cats; on return to the control diet, endotoxin
concentrations increased to those characteristic of the
baseline (Figure 2).
Discussion
Dietary supplementation with L acidophilus
DSM13241 resulted in various health-related effects
host. A variety of changes were observed in
the commensal gastrointestinal tract flora
and health or immune status of cats in
response to probiotic administration. The
probiotic was recovered from feces of cats
demonstrating survival through the feline
gastrointestinal tract. Persistence of the pro-
biotic, however, appeared to cease following
the withdrawal of supplementation.
Changes associated with the probiotic
feeding phase alone and persisting follow-
ing withdrawal of the probiotic were
observed in association with the probiotic
L acidophilus DSM13241. Persistence for
the period following withdrawal of the pro-
biotic was largely observed for
immunomodulatory factors altered during
facilitated by colonization at numbers
below the detectable limit. The exception to this was
blood endotoxin concentrations, which were reduced
only in the probiotic-feeding phase, consistent with a
direct mode of action such as enhanced gastrointestinal
barrier function or reduced lumenal bacterial endotox-
in. Conversely, changes in gastrointestinal bacterial
populations were largely associated with the supple-
mentation phase, with fewer measurements or those of
lower magnitude providing evidence of a continued
effect on the gastrointestinal tract.

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Confounding effects as a result of adaptation to
environmental factors or diet, although not impossible,
are considered unlikely. All cats were maintained in the
same environmentally enriched housing as that used
during the study and had previously been fed nutri-
tionally complete commercial dry pet food of similar
composition to the base diet. The additional adaptation
to base diet for 3 to 5 weeks prior to collection of feces
and 5 weeks prior to obtaining hematologic measure-
ments makes this increasingly unlikely.
A combination of culture-based and molecular
identification methods were used in assessment of the
gastrointestinal microflora to counteract inaccuracies
inherent in the use of selective culture alone to inves-
tigate the microflora of companion animals.20,26 Some
evidence of this inaccuracy was observed in our study
where numbers of lactobacilli remained unchanged as
measured by bacterial culture while FISH revealed
increased numbers of lactobacilli and L acidophilus
during probiotic supplementation. Selective bacterial
culture is reliant on the provision of optimal growth
conditions (which include temperature and nutrient
and atmospheric factors) for target bacterial popula-
tions within the complex gastrointestinal microflora.
Furthermore, culture media are largely developed for
the selection of human isolates adding to these inade-
quacies. Because FISH uses specific DNA probes that
hybridize the 16S rRNA, the technique can be used to
detect viable, yet nonculturable, bacteria on the basis
of 16S rRNA nucleotide sequence.
Enumeration of enterococci, E faecalis, and
Clostridium spp by FISH and selective culture revealed
the techniques to be largely similar in describing the
effect of probiotic feeding on these populations.
Bacterial groups enumerated were selected as markers
of microflora health with Clostridium and Enterococcus
spp representing detrimental bacterial markers and lac-
tobacilli and Bifidobacterium spp representing benefi-
cial species. In contrast to the health-promoting short-
chain fatty acids produced by lactic acid bacteria, the
proteolytic nature of Clostridium spp results in the pro-
duction of putrefying metabolites. Clostridium perfrin-
gens, C difficile, and E faecalis are also considered
opportunistic pathogens, the former 2 being associated
with gastrointestinal infection and diarrheal disease
and the latter with wound and systemic infections in
companion animals.33,34 The observed reductions in the
clostridia and E faecalis populations, combined with
increased numbers of lactobacilli and L acidophilus
observed by FISH, are highly suggestive of a healthier
balance of the gastrointestinal microflora occurring
during probiotic supplementation. The reduction in
Bifidobacteria as a percentage of the total population is
not considered to counteract the suggestion of a
healthier balance. The magnitude of change in this
beneficial population was low, and the population was
comparatively lower in number than the Lactobacillus
population, suggesting a bias towards the latter in the
healthy feline gastrointestinal tract.
The FISH-mediated detection of the probiotic
species involved the use of a probe specific for a sub-
group of lactobacilli comprising L acidophilus, L crispa-
tus, L gallinarum, and L helveticus. Low numbers of
1010
these species observed in the baseline period, com-
pared with the probiotic phase, indicate that an
increase in the probiotic strain occurred. However,
stimulation of L crispatus, L gallinarum, and L helveti-
cus populations during probiotic feeding cannot be dis-
counted. The increased numbers of lactobacilli
observed during probiotic feeding exceed those detect-
ed by the general Lactobacillus probe. It is therefore
possible that feeding of the probiotic strain stimulated
numbers of the inherent Lactobacillus populations.
The reduction in fecal pH likely reflects the
observed changes in the microflora, such as increased
numbers of lactic acid bacteria. The mechanism of per-
sistence in fecal pH reduction is unclear; however,
decreased colonic pH is considered a key factor in the
effect of probiotic bacteria. This observation may
therefore suggest an environment favoring the growth
of beneficial species and inhibiting detrimental gas-
trointestinal tract species.35
Probiotic bacteria, particularly Lactobacillus spp,
have previously been shown to modulate nonspecific
immune reactions in humans and other animals.20,22,36
The immunomodulatory effect of L acidophilus
DSM13241 in cats was apparent in several parameters
measured in the current study. All systemic changes in
this healthy population, however, were relatively small
in magnitude, and measured values were within refer-
ence range.
Immunomodulation was suggested by altered lym-
phocyte and eosinophil populations with reduced and
increased numbers, respectively, throughout the study
and by increased activity of peripheral blood phago-
cytes. Small changes in the phagocytically active pro-
portion of granulocytes and monocytes occurred
across the course of the study. The granulocyte popu-
lation, however, had substantially increased numbers
of fluorescent-labeled E coli phagocytosed per cell
when challenged in vitro. Furthermore, this increase
was observed in a background of stable granulocyte
(neutrophil) numbers. These data suggest that L aci-
dophilus administration in cats may enhance the
phagocytic capacity of circulating granulocytes and
that the effect is maintained at least 4 weeks after the
cessation of probiotic feeding. Because granulocytes
account for approximately 50% of the WBC population
in healthy adult cats, the phagocytic capacity of
peripheral blood is almost entirely the result of the
stimulated population. The nonspecific immune stim-
ulation observed in the postprobiotic period, therefore,
involves newly developed or maturing cells not direct-
ly influenced by probiotic feeding. It is not known
whether the prolonged nature of this effect is the result
of the nature of the immunomodulatory mechanism or
because of continued presence of the probiotic in the
gastrointestinal tract at levels below the detection
limit. Different Lactobacillus doses are known to be
required for immune stimulation, compared with fecal
colonization, with the former occurring at compara-
tively lower doses in human studies.36
Effects on general health were observed in cats in
response to probiotic feeding, with blood endotoxin
concentrations reduced during probiotic feeding and
improved resistance to osmotic lysis in RBCs over the
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