Curr Microbiol DOI 10.1007/s00284-014-0610-z Viable Intestinal Passage of a Canine Jejunal Commensal Strain Lactobacillus acidophilus LAB20 in Dogs
Yurui Tang• Per E. J. Saris
Received: 3 March 2014 / Accepted: 27 March 2014
Ó Springer Science+Business Media New York 2014
Abstract The strain Lactobacillus acidophilus LAB20
with immunomodulatory properties was previously found
dominant in the jejunal chyme of four dogs, and the novel
surface layer protein of LAB20 suggested its competitive
colonization in canine gut. To evaluate the persistence and
survival of LAB20 in healthy dogs, LAB20 was fed to five
healthy pet dogs for 3 days, at a dosage of 108 CFU daily as
fermented milk supplement. The fecal samples, from 1 day
prior to feeding, three continuous feeding days, and on day
5, 7, 14, and 21, were collected for strain-specific detection
of LAB20 using real-time PCR. We found that LAB20
count was significantly increased in dog fecal samples at
the second feeding day, but rapidly decreased after feeding
ceased. The fecal samples from prior to feeding, during
feeding, and post-cessation days were plated onto mLBS7
agar, from where LAB20 was recovered and distinguish-
able from other fecal lactobacilli based on its colony
morphotype. Using strain-specific PCR detection, the col-
onies were further verified as LAB20 indicating that
LAB20 can survive through the passage of the canine
intestine. This study suggested that canine-derived strain
LAB20 maintained at high numbers during feeding, viably
transited through the dog gut, and could be identified based
on its colony morphotype.
Introduction
In the gastrointestinal tract (GIT) of vertebrates, a dynamic
and sophisticated balance of microbial ecology is
Y. Tang P. E. J. Saris (&)
Department of Food and Environmental Sciences, University of
Helsinki, P. O. Box 56, 00014 Helsinki, Finland
e-mail: Per.Saris@helsinki.fi
established beginning at birth and continues to develop
throughout the life span of the host. The variation in host
dietary preference, age, lifestyle, and genetic background
shapes microbiota to be specific among individuals [5, 8,
31]. The microbiota inhabits the GIT, in turn, facilitates
nutrient digestion, and interacts with host by mediating
immune responses. Lactobacillus species are present
throughout the mammalian GIT [12, 36], and some Lac-
tobacillus strains are prevalently utilized in probiotic pro-
ducts. Probiotics are suggested to promote the health of
human and also animals, by preventing pathogen invasion,
producing antimicrobial substances, and enhancing the
immune responses [10]. Recent clinical studies have shown
that probiotics may be helpful to prevent or treat a variety
of acute and chronic gastrointestinal diseases, such as
antibiotic-associated diarrhea and inflammatory bowel
diseases [7, 24]. For companion animals, probiotics have
also been recommended to alleviate gut problem led by
intestinal dysbiosis [3, 37]. However, most canine probiotic
products contain human-derived strains [37]. With respect
to host specificity, probiotic strains with sufficient capa-
bility to adapt and colonize to the canine gut are needed.
Probiotics are typically transient residents in the intes-
tine, although probiotic strains show the ability to tolerate
and colonize the intestine both in vitro and in vivo [1, 11,
35, 38]. Therefore, it is intriguing to investigate how and
why endogenous bacteria could maintain themselves in the
GIT, and it may shed light to probiotic applications. There
have been a number of studies investigated the probiotic
effects of strains isolated from canine origin [2, 16–18, 21,
25]; however, most of those studies are confined to canine
fecal isolates. There is increasing number of supporting
evidence for that fecal microbiota differs from that in the
upper intestine [14, 19, 39]. O’Mahony et al. [20] isolated
commensal strains with probiotic potential from the large
123Y. Tang, P. E. J. Saris: Viable Intestinal Passage of Lactobacillus acidophilus LAB20
intestine of healthy canines, but only Bifidobacteria ani-
malis AHC7 was involved in the intervention study, which
reduced the carriage of Clostridia in dogs. To our knowl-
edge, there has been no intervention studies with Lacto-
bacillus strains derived from canine intestinal tract.
Previously, Tang et al. [29] presented the dominance of
L. acidophilus LAB20 in canine jejunal chyme of fistulated
dogs. Four out of five dogs harbored endogenous LAB20 as
dominant lactobacilli strain suggesting that this particular
strain could be a good candidate to investigate the possible
factors facilitating it to colonize and be predominant in dog
gut. Recently, LAB20 was found able to attenuate LPS-
induced interleukin-8 in HT-29 cells, suggesting the
potential probiotic properties of LAB20 to alleviate intes-
tinal problems associated with inflammation [30]. In the
present study, one aim was to evaluate if fed LAB20 can
persist in canine gut of healthy dogs, as suggested by Tang
and Saris’ previous study [28]. Another aim was to study if
endogenous LAB20 can survive the passage of the canine
intestine.
was stored at 4 °C before feeding, enumerated and exam-
ined by microscopy before delivery to the dog owners.
Feeding and Sample Handling
LAB20 was fed to dogs in fermented milk (3.4 9
106 CFU/ml, 50 ml per meal) with dog food. Five healthy
pet dogs (from 3 to 10 years old) from different owners
(breeds included Collie Smooth, Siberian husky, Welsh
corgi Pembroke, and Dogo argentino) were fed with
LAB20 supplementary for 3 days. Fresh fecal samples
were collected into a sterile Falcon tube after defecation
and stored in-20 °C. Samples were collected from 1 day
prior to LAB20 feeding and 3-day feeding period followed
by four collections at day 5, 7, 14, and 21. The sample
collection dates were chosen based on the results of the
previous study [28], and a shorter feeding period was
chosen because LAB20 showed highest copy numbers in
dog feces at feeding day 3 in the previous study. The study
was approved by the University of Helsinki ethics
committee.
Materials and Methods
Freeze Drying of LAB20 and Fermented Milk
Preparation
Lactobacillus acidophilus LAB20 was previously isolated
from canine jejunal chyme [29]. It was cultured in LBS
broth aerobically (BBL, Becton–Dickinson Microbiology
System, Cockeysville, MD, USA) without acetic acid and
pH adjusted to 7 with 5 M NaOH (mLBS7) to optimize
LAB20 growth.
Due to rather poor growth of LAB20 (106-7 CFU/ml in
mLBS in 18 h), LAB20 cells were concentrated and stored
as freeze-dried batches. For each batch of freeze-dried
bacteria, 400 ml mLBS7 broth was inoculated with over-
night LAB20 culture (1 % inoculum), and grown at 37°C
for 18 h. Bacterial cells were harvested by centrifugation at
16,000g for 10 min at 4 °C and washed twice with ice-cold
0.85 % NaCl. Bacterial cells were then resuspended in
5 ml of cryoprotectant (11 % w/v skim milk powder, 1 M
glycerol, 10 % w/v trehalose), five aliquots were let to
stand in room temperature for 10 min allowing equilibra-
tion between cells and cryoprotectant, and then lyophilized
in a Hetovac VR-1 (HETO Lab Equipment, Denmark).
Viability of bacteria was determined before and after
freeze-drying by serial dilution, and plated on mLBS7 plate
and incubated at 37 °C for 48 h. Freeze-dried LAB20 cells
were stored at 4 °C.
Approximately 107 CFU of LAB20 were inoculated into
50 ml sterilized (110°C 15 min) skimmed milk (10 %
w/v), and incubated at 37°C for 25 h. The fermented milk
DNA Extraction and Real-Time PCR Detection
Total genomic DNA of fecal samples was extracted using
QIAamp DNA stool kit (Qiangen Inc., Valencia, CA,
USA). The strain-specific real-time PCR detection was
performed according to the methods described by Tang and
Saris [28]. Briefly, serial dilutions of pLEB767, plasmid
carrying partial S-layer protein gene of LAB20, were used
for calibration. Primers RT1 and RT2 were designed to
specifically target 136 bp variation region of LAB20
S-layer gene. Real-time PCR was performed with Maxima
SYBR Green/ROX qPCR Master Mix (Thermo Scientific,
Helsinki, Finland) on the ABI Prism 7300 (Applied Bio-
systems, Foster City, CA).
Plate Fecal Samples and PCR Detection
According to real-time PCR results, the fecal samples with
highest LAB20 counts, together with the samples prior to
feeding, day 7, and day 21 samples from each dog (dog 2
did not defecate at day 21), were diluted with saline water
and plated on mLBS7 agar plates. After incubation at
37 °C for 2 days, the colony morphotype was inspected
and compared to LAB20. The colonies with similar mor-
photype to LAB20 were randomly picked and checked by
PCR, targeted to variable region of LAB20 S-layer protein.
In order to investigate the freeze and defreeze procedure on
the LAB20 viability, aliquot fecal sample at pre-feeding
day was spiked with LAB20 and plated with or without
freezing treatment.
123Y. Tang, P. E. J. Saris: Viable Intestinal Passage of Lactobacillus acidophilus LAB20
Fig. 1 Fecal LAB20 counts detected with strain-specific PCR
targeted to variable region of L. acidophilus LAB20 S-layer protein
gene. LAB20 fermented milk was fed (1.7 9 108 CFU daily) to dogs
from day 1 to day 3, and fecal samples were collected prior to feeding
and at days 1, 2, 3, 5, 7, 14, and 21. Dog 5 did not defecate at day 1,
and samples from dog 3 at day 14 and 21 could not be obtained from
owner. Data are generated from three fecal samples, respectively, and
expressed as means ± standard deviations
Statistical Analysis
Logarithms of fecal partial S-layer protein gene copy
numbers were used to achieve distribution of LAB20, and
analyzed using SPSS (IBM SPSS statistics 21) with one-
way ANOVA with time as the main factor; differences
among means of dogs were analyzed by the Student–
Newman–Keuls test. Differences were considered statisti-
cally significant at P \ 0.05.
Results
LAB20 Counts in Freeze-Dried Batch and Fermented
Milk
Due to rather poor growth, LAB20 cells were concentrated
and stored as freeze-dried batches. Approximately 107
CFU of LAB20 were inoculated into 50 ml sterilized
skimmed milk (10 % w/v), after 25 h incubation, the fer-
mented milk contained 3.4 9 106 CFU/ml LAB20. In
order to test the survival of LAB20 during storage, fer-
mented milk was stored at 4 °C for three days, the CFU of
LAB20 in fermented milk were counted daily. The result
showed that LAB20 counts remained nearly the same
during three days storage (data not shown).
Real-Time PCR Detection of LAB20 in Dog Fecal
Samples
To determine the LAB20 counts in dog feces during the
intervention study, strain-specific real-time PCR was used
targeting the variation region of LAB20 S-layer protein
gene. The gene copy number of LAB20 prior to the feeding
was set as the baseline for real-time PCR detection, and it
varied from 0 ± 0 to 102.98 ± 100.22 copies/g among
individual dogs. During the feeding period, LAB20 was
detected from fecal samples with significantly increased
numbers at day 2 and 3 compared to the following col-
lection days and prior to feeding (P \ 0.01). After the
feeding ceased, LAB20 counts were back to initial baseline
gradually except dog 1, in which LAB20 counts dropped to
baseline after second feeding day (Fig. 1). There was no
significant (P [ 0.05) difference among individual dogs
concerning the changing trend of LAB20 counts during the
collection period.
Storage and melting process could have lethal effect on
LAB20, which may lead to bias of the actual LAB20
counts in dog feces. Therefore, LAB20 was spiked to fecal
samples with or without freeze storage to study the effect
of storage and thawing on LAB20 in dog feces. Two ali-
quot fecal samples were spiked with 200 ll of LAB20
overnight culture. Without freezing and melting 3.15 9
106 CFU/g of LAB20 was obtained. Freezing at-20 °C
for two days and melting the sample before plating yielded
1.34 9 105 CFU/g LAB20.
LAB20 Colonies on Fecal Sample Plates
To determine if LAB20 can survive through canine gut, the
fecal samples at prior feeding, the day with highest LAB20
counts with real-time PCR, and samples at day 7 and 21
were plated on mLBS7 agar. By comparing with the prior
to feeding samples, the colony with unique morphotype
and similar to LAB20 was recognized from every feeding
123Y. Tang, P. E. J. Saris: Viable Intestinal Passage of Lactobacillus acidophilus LAB20
Fig. 2 Colonies formed on
mLBS7 agar from fecal samples
at same dilution (feces diluted
1049). a Colonies from fecal
sample of prior feeding day of
dog 2, and the colonies are
magnified in b. c Colonies from
fecal sample of feeding day 3 of
dog 2, and the colonies are
magnified in d (from further
tenfold dilution of the same
fecal sample, to give clear
image of colonies)
Fig. 3 PCR-amplified partial LAB20 S-layer protein gene from fecal
colonies. Lanes 1–8 fecal colonies from samples of dog 1. Lanes 9–17
fecal colonies from dog 2. Lanes 18–24 fecal colonies from dog 3.
Lanes 25–34 fecal colonies from dog 5. Lanes 35–41 fecal colonies
from dog 4. Lanes 42 is LAB20 as positive control. Underline symbol
‘‘
-’’ indicates the morphotype of colony on plate is different than
LAB20, whereas ‘‘?’’ represent the fecal colony morphotype similar
to LAB20 colony
123Y. Tang, P. E. J. Saris: Viable Intestinal Passage of Lactobacillus acidophilus LAB20
period plate of each dog. The colonies in prior to feeding
samples were replaced or outnumbered by LAB20-like
colonies from each dog feces samples of LAB20 feeding
day 2. The unique morphotype colony was rather flat and
with rough surface relative to other lactobacilli which were
less flat and had moist and smooth surface (Fig. 2). Colo-
nies from dog 1 are presented (Fig. 2), other plates were
found with similar results (not shown). To verify if the
unique morphotype colonies were LAB20, strain-specific
PCR was conducted. The colonies with different morpho-
types gave no amplification, whereas these unique colonies
yielded amplification with same size as LAB20 (Fig. 3).
Discussion
Lactobacilli are widely applied as probiotics to pursue its
desired beneficial effects on the hosts. Although many
probiotic strains present sufficient adhesion ability in vitro,
which is usually considered to contribute to the coloniza-
tion and persistence in the GIT, many of them fail to persist
in the GIT for long period [9, 15, 20, 38]. However,
identifying a probiotic with a reasonable persistence period
could represent a significant development, not only because
it could reduce the frequency of ingestion, but also because
it implies that the strain can adapt and thrive in the GIT.
Most probiotic strains and candidates are isolated from
fecal samples, but fecal isolates may just be transients
which fail to compete with endogenous inhabitants and
establish themselves in GIT. Furthermore, fecal isolates
mainly represent the microbiota in distal gut rather than
proximal gut [13, 19]. It has been shown that the pre-
existing microbial ecosystem is rather stable and resilient,
as it has been coevolved with the host over time [34].
Microbiome differences exist among individuals, due to
individual genetic background, immune response, and
dietary preferences [4, 22, 23, 26, 32], however, the met-
abolic end products are typically quite similar [32].
Therefore, a stable ecosystem is maintained as long as
microbial members of the community existing in GIT are
able to perform similar functions [6, 27]. This may explain
why it is strenuous for one single or a combination of
probiotic bacterial strains to integrate with pre-existing
ecosystem in the gut. Hence, it is intriguing to investigate
how the endogenous bacteria could maintain in the GIT,
especially the predominant ones, and may shed light on
probiotic applications.
Lactobacillus acidophilus LAB20 was previously found
predominant in canine jejunal lactobacilli, showed poten-
tial to persist in canine GIT for over six weeks post-
administration, and recently found with immunomodula-
tory properties [30]. Therefore, it was employed in the
present study to investigate its potential to persist and
transit through dog gut [29]. In this study, strain-specific real-
time PCR was used to detect LAB20 in dog feces, which was
previously validated [28]. Within three feeding days, LAB20
was detected with significantly (P \0.01) increased amount
in fecal samples, whereas dropped to baseline after feeding
ceased. The result suggests that LAB20 could transit through
dog gut during administration period. On the other hand, the
absence of LAB20 in following days indicated that either
LAB20 was unable to persist in canine gut, or it was diluted
to an undetectable amount in the feces, or it remained in the
canine gut and did not passage. Without access to the biopsy
samples, this could not be evaluated. However, in the study
of Valeur et al. [33], L. reuteri community in the stomach and
duodenum was found bigger than in fecal samples after
administration of human-derived L. reuteri ATCC 55730.
Further, by plating biopsy samples, no live L. reuteri were
found recovered on plates [33]. In another administration
study, Alander et al. [1] found that human-derived
L. rhamnosus GG generated from fecal samples may
underestimate colonization of probiotic strains in vivo.
Therefore, to determine whether LAB20 could colonize in
canine gut further investigation with biopsy samples is
needed.
The viability of LAB20 after transit through dog gut was
studied with plating assay. As the fecal samples had been
stored at-20 °C, the lethal effect of storage was first
determined. The result showed that the viability of LAB20
in feces reduced 20-fold during freezing and thawing
process. By plating fecal samples on mLBS7 agar, LAB20
was recovered from samples at feeding day which showed
abundant counts in real-time PCR, indicating LAB20 can
survive passage through dog gut (about 104–106 CFU/g of
feces). Interestingly, LAB20 colonies could be easily dis-
tinguished from that of other lactobacilli on mLBS7. By
comparing with the prior feeding samples, colonies with
unique morphotype similar to LAB20 were recognized
from each dog and further confirmed by LAB20-specific
PCR detection. Identification and quantification of LAB20
by colony morphotype inspection provides an easier way
than LAB20-specific real-time PCR to detect LAB20 sur-
viving the intestinal passage in future studies.
In conclusion, canine endogenous strain L. acidophilus
LAB20 was found able to survive at high numbers through
canine GIT during administration, and its colonies have
unique and distinguishable morphotype grown on the
mLBS7 agar plates. This feature of LAB20 facilitates the
future probiotic intervention studies, as its presence can be
monitored directly upon plating feces in the preliminary
inspections.
Acknowledgments We thank volunteers for providing dogs and
collecting fecal samples, Tuomas Puukko for kindly help to take the
bacterial colony photographs, and Dr. Timo M. Takala for his help in
123Y. Tang, P. E. J. Saris: Viable Intestinal Passage of Lactobacillus acidophilus LAB20
revision of this article. The authors would like to thank Viikki
Graduate School in Biosciences (VGSB) for study support, and
financial support from China Scholarship Council (CSC).
Conflict of interest of interest.
The authors declare that they have no conflict
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