97
Introduction
Probiotics are live microorganisms
that when administered in adequate
amounts confer a health eect on the
host.
1
There have been many studies of
the eects of probiotics on the health
of humans, but few in small animals. In reviews of human
studies involving probiotics. it was stated that well-established
probiotic eects include:
1,2
1. Prevention and/or reduction of duration and complaints
of rotavirus-induced or antibiotic-associated diarrhea as
well as alleviation of complaints due to lactose intolerance;
2. Reduction of the concentration of cancer-promoting
enzymes and/or putrefactive (bacterial) metabolites in
the gut;
3. Prevention and alleviation of unspecic and irregular
complaints of the gastrointestinal tracts in healthy people;
4. Benecial eects on microbial aberrancies, inammation
and other complaints in connection with inammatory
diseases of the gastrointestinal tract, Helicobacter pylori
infection or bacterial overgrowth;
5. Normalization of passing stool and stool consistency in
subjects suering from obstipation or an irritable colon;
6. Prevention or alleviation of allergies and atopic diseases
in infants; and
7. Prevention of respiratory tract infections (common cold,
inuenza) and other infectious diseases as well as treat-
ment of urogenital infections.
Immune-mediated and infectious diseases are very com-
mon in small animals so the potential benecial eects of
probiotics that can immune modulate could signicantly
impact veterinary practice. All mechanisms of immune modu-
lation have not been characterized, and it is clear that these
eects vary by the probiotic. Some probiotics may benecially
inuence innate and acquired immunity systemically by a
variety of proposed mechanisms including inducing cytokine
production, natural killer cell activity, and both specic and
nonspecic immunoglobulin production.
2
Several review articles in human medicine have recently
suggested that the evidence indicating probiotics have provided
a benecial eect for human conditions such as Clostridium
dicile diarrhea and hospital-acquired pneumonia is minimal
and that larger, more rigorously controlled multicenter studies
should be performed. These ndings emphasize that biological
eects of individual probiotics will vary and that each pro-
biotic introduced should be rigorously
evaluated in a controlled fashion to
dene the potential for clinical utility.
3-5
In addition, the source of the probiotic
should also be considered. For example,
in recent veterinary studies, the majority
of products claiming to contain probiotics generally did not
meet the label claim when evaluated.
6,7
One exception is the
Purina Pro Plan Veterinary Diets probiotic, Enterococcus
faecium SF68 (FortiFlora). It was recently shown that this
probiotic stored appropriately still met the label claim for
bacterial numbers several years aer production.
The potential benet of immune-modulating probiotics to
animal health could be considerable.
8
Some of the strongest
data supporting immune-modulating properties of some
probiotics in people and dogs are associated with the treat-
ment of atopy and inammatory bowel diseases.
9-13
Enterococcus faecium strain SF68 (NCIMB10415) was
originally isolated from the feces of a healthy baby and was
initially shown to inhibit the growth of a number of entero-
pathogens.
14
The purpose of this lecture is to summarize
key ndings regarding past and ongoing studies of the
potential immune-modulating eects of this probiotic.
Dog Immune Modulation Studies
Enterococcus faecium strain SF68 was fed to a group of
puppies vaccinated for canine distemper virus (CDV) and
compared over time to a control group that was similarly
vaccinated, but not fed the probiotic.
15
A number of ndings
suggested an immune-modulating eect of the probiotic.
The puppies supplemented with SF68 had increased serum
and fecal total IgA concentrations, increased CDV-specic
IgG and IgA serum concentrations, and increased percent-
age of circulating B lymphocytes when compared to control
puppies. The eect on CDV-specic IgG and IgA antibodies
in serum was seen only aer the puppies had been sup-
plemented for 31 and 44 weeks, and it was believed that
SF68 prevented the decline in antibody titers observed in
the controls by maintaining high levels of antibodies in the
supplemented puppies.
In the rst study, immunological parameters were not
assessed until 10 weeks aer starting supplementation with
the probiotic. Clinical observations suggest that immune
modulation induced by this probiotic may occur sooner than
10 weeks. Thus, a double-blind, placebo-controlled study
Evidence for Immune Modulation Induced by a Probiotic
Michael Lappin, DVM, PhD, DACVIM
Colorado State University
College of Veterinary Medicine and Biomedical Sciences
Fort Collins, CO
michael.lappin@colostate.edu
Glossary of Abbreviations
CDV: Canine Distemper Virus
IFA: Immunofluorescent Anti-
body Testing
98
in healthy young adult Beagles was recently performed at the
Center for Companion Animal Studies. Due to prepublication
restrictions, this proceedings will only present the general
ndings with more extensive review of the results presented
at the Summit. Using ow cytometry for the measurement
of cellular ndings and a serum-based cytokine panel,
evidence for immune modulation induced by the probiotic
was shown as early as four weeks aer supplementation.
16
In another ongoing study at the Center for Companion
Animal Studies, the eect of E. faecium SF68 on the clinical
outcomes of American Pitbull Terriers with generalized
demodecosis will be shared at the Summit.
Kitten Immune Modulation Study
In a follow-up study, a similar experimental design used
to assess vaccine responses in puppies was applied to a study
group of kittens.
17
In that study, it was hypothesized that
feeding E. faecium SF68 to kittens would enhance nonspecic
immune responses, FHV-1, FCV, and FPV-specic humoral
immune responses, and FHV-1-specic cell-mediated immune
responses of kittens. Twenty 6-week-old SPF kittens were
purchased from a commercial vendor and divided into two
groups. One group was fed SF68 daily, and the other group
was fed the placebo starting at 7 weeks of age.
At 9 and 12 weeks of age, a commercially available FVRCP-
modied live vaccine was administered SQ, and the kittens
were followed until 27 weeks of age. The attitudes and
behavior of the kittens were monitored daily throughout the
study. Body weight was measured weekly. Blood, saliva, and
feces were collected from all cats prior to starting probiotic
or placebo supplementation at 7 weeks of age and at 9, 15,
21, and 27 weeks of age. In addition, feces were collected
from kittens in the treatment group aer the study was
completed at 28 weeks of age.
For each group of kittens, ve fecal samples per day were
randomly selected from the shared litter box and scored
using a standardized graphic scoring card. Fecal extracts
from samples taken at 9 and 27 weeks of ages were analyzed
for total IgA and total IgG. Other parameters monitored
included randomly amplied polymorphic DNA (RAPD)-PCR
on feces to determine if viable E. faecium SF68 was in the
stools of treated cats and to assess whether the probiotic was
accidentally transmitted from the treated kittens to the
control kittens. Commercially available ELISAs were used to
determine whether Clostridium perfringens enterotoxins or
C. dicile toxins A/B were present in the feces of the kittens.
Routine aerobic fecal cultures for Salmonella spp. and
Campylobacter spp. were performed. Complete blood counts,
serum biochemical panels, and urinalyses were performed to
assess for adverse events induced by the probiotic. Antigen-
specic humoral immune responses were estimated by
measuring serum FHV-1-specic IgG, FHV-1-specic IgA,
FCV-specic IgG, and feline panleukopenia-specic IgG
in sera as well as FHV-1 specic IgG and IgA levels in saliva
using adaptations of previously published ELISA assays.
Total IgG and IgA concentrations in sera, fecal extracts,
and saliva were estimated by use of commercially available
ELISA assays or radial immunodiusion assay. Cellular
immune responses were assessed via ow cytometry and
whole blood proliferation assays. Lymphocytes were stained
for expression of CD4, CD8, CD44, MHC Class II, and B cells.
In addition, lymphocyte proliferation in response to conca-
navalin A and FHV-1 antigens was assessed.
Body weight and fecal scores were not statistically dierent
between the two groups over time or at any individual time
points. Feces from seven of nine treatment cats were positive
for SF68 on at least one time point during the study, whereas
feces from all control cats were negative for SF68 at all time
points. SF68 DNA was not detectible from feces of any treated
cat one week aer stopping supplementation (week 28).
All samples from placebo cats were negative for SF68 by
RAPD-PCR. Neither Salmonella spp. nor Campylobacter spp.
were grown from feces. Numbers of positive samples for
C. dicile toxins A/B or C. perfringens enterotoxin were not
signicantly dierent between the groups over the course
of the study.
Complete blood counts and biochemical proles were
within normal limits for the age group for all cats at all time
points. A number of the immune markers were numerically
greater in the SF68 kittens versus the placebo group, but did
not reach statistical signicance. For example, at 21 and 27
weeks of age, the mean levels of FHV-1-specic IgA in serum
and saliva were greater in the treatment group when compared
to the placebo group. Moreover, the mean FHV-1-specic
serum IgG levels were greater in the treatment group when
compared to the placebo group at 15, 21, and 27 weeks of age.
At 15 weeks of age, the treatment group serum mean FPV-specic
IgG levels were greater than those of the placebo group.
There were no statistical dierences between the groups
for any cell surface markers at the rst four time points.
However, at 27 weeks of age, the treatment group had a
signicantly higher percentage of gated lymphocytes pos-
itive for CD4 (mean 13.87%) than the placebo group (mean
10.61%, p = 0.0220).
In this study, we concluded that SF68 was safe to admin-
ister to cats and the increase in CD4+ cell counts in the
treatment group compared to the placebo group without
a concurrent increase in CD8+ counts at 27 weeks of age
demonstrated a systemic immune modulating eect by the
probiotic. Because we did not show a signicant increase
in lymphocyte stimulation by FHV-1 or an increase in the
expression of the memory cell marker CD44 on the CD4+
lymphocytes in the treatment group, the increase in CD4+
T lymphocytes may have been nonspecic as the cells
appeared to be unprimed. As the CD4+ T lymphocytes of
kittens in this study were not additionally characterized via
cytokine production proles or additional cell-surface
marker characterization; it could not be determined whether
99
a Th1 or Th2 response predominated. We believed that sample
size and/or the duration of the study may have precluded
detection of statistical dierences between the groups in
regard to FPV, FCV, and FHV-1 antibody titers.
Chronic Feline Herpesvirus 1 Study
Since the normal kitten study documented potential eects
of E. faecium SF68 on cell-mediated immunity in cats, we
chose to study the potential eects on feline herpesvirus 1
(FHV-1).
18
This infectious agent is extremely common in cats
and is frequently associated with morbidity because of
recurrent ocular and respiratory clinical signs of disease.
In addition, there is no known drug therapy that consistently
eliminates the carrier state, and vaccination does not provide
sterilizing immunity. In this study, it was hypothesized that
feeding SF68 would decrease clinical disease, episodes of
FHV-1 shedding, and numbers of FHV-1 DNA copies shed
over time in cats with chronic FHV-1 infection.
11
Overall, 12 cats with chronic FHV-1 infection were admin-
istered either SF68 or the palatability enhancer as a placebo,
monitored for clinical signs of disease, monitored for FHV-1
shedding, and evaluated for FHV-1-specic humoral and
cell-mediated immune responses as well as for fecal microbi-
ome stability. Aer an equilibration period, mild stress was
induced over time by changing the housing of the cats from
cages to group housing multiple times over a ve-month
period.
The SF68 was well-tolerated by all cats. Fecal microbial
diversity was maintained throughout the study in cats sup-
plemented with SF68, but decreased in cats fed the placebo,
indicating a more stable microbiome in cats fed SF68. Upper
respiratory signs of disease were not exacerbated in this model
of stress. While results varied among cats, those administered
SF68 had fewer episodes of conjunctivitis than the placebo
group during the supplementation period suggesting that
administration of the probiotic lessened morbidity associated
with chronic FHV-1 infection exacerbated by stress.
Murine Acute Giardia Study
In previous work, mice administered SF68 and then infected
with Giardia intestinalis shed fewer trophozoites and less
Giardia antigen than the placebo group.
19
In addition, supple-
mented mice had increased CD4+ cells in Peyer’s patches
and the spleen as well as increased anti-Giardia intestinal
IgA and serum IgG when compared to untreated mice.
Chronic Subclinical Giardia Study in Dogs
When SF68 was administered to 10 adult dogs with chronic,
subclinical Giardia infection, no dierences in cyst shedding
or fecal antigen testing were found when compared to 10
placebo-treated dogs.
20
In addition, there were no dierences
between groups in fecal IgA concentrations. In contrast to the
mouse study, the dogs were previously infected by Giardia,
which may have aected the results. In addition, the study
was only for six weeks; in the previously discussed puppy
study, some of the signicant immune-modulating eects
were not seen until later in the supplementation period.
1
Shelter Animals Acute Nonspecific
Diarrhea Study
In a recent study, we hypothesized that cats and dogs
housed in an animal shelter that were fed SF68 would have
decreased episodes of diarrhea and improved fecal scores
compared to untreated cats and dogs in the same environ-
ment.
21
The cats were housed in two dierent rooms, and
the dogs were housed in two dierent rooms in a northern
Colorado Animal Shelter. The cats and dogs were all fed
a standardized diet by species. Animals in one room were
supplemented daily with E. faecium SF68, and animals in
the alternate room were supplemented daily with a placebo.
Otherwise, management of the rooms was identical for the
duration of the study. To reduce risk of a room inuence on
the results of the study, the room in which cats or dogs were
being supplemented with E. faecium SF68 was switched
aer one month, with a one-week washout period to lessen
the possibility that SF68 surviving in the environment could
inuence the results of the study.
During the study, routine shelter cleaning and disinfection
protocols were being followed. Prior to cleaning the room
each morning, feces in the cage of each animal was scored
by one of the investigators using the Purina Fecal Scoring
System for Dogs and Cats. This person was blinded to the
treatment groups. Aer scoring, feces from dogs with a score
from 4 to 7 (indicating mild to severe diarrhea) were collected
and transported to Colorado State University for infectious
disease testing, which included microscopic examination
for parasites eggs, cysts, and oocysts aer zinc sulfate
centrifugation otation and immunouorescent antibody
testing (IFA) for Cryptosporidium oocysts and Giardia cysts
(Meriuor
®
Cryptosporidium/Giardia, Meridian Bioscience
Inc., Cincinnati, OH). The percentages of dogs or cats with
diarrhea of >2 days duration were calculated over the course
of the study. A generalized linear mixed model using a
bionomial distribution with treatment being a xed eect
and the room being a random eect was used to assess for
statistical dierences between treatment groups. Presence
of parasites was included as a covariate. Signicance was
dened as p < 0.05.
Diarrhea prevalence rates were low for all dogs in the study,
and statistical dierences were not detected. However, the
percentage of cats with diarrhea >2 days was 7.7% for the
probiotic group and 20.7% for the placebo group. This result
was signicantly dierent (p = 0.0297). These results suggest
that administration of SF68 to cats housed in shelters may
lessen the numbers of days with diarrhea. As this was a
short-term study, this eect may have been from probiotic
inuences on intestinal microbiota rather than systemic
immune enhancing eects.
100
Summary and Conclusions
The evidence gathered to date suggests that E. faecium SF68
has immune-modulating eects in dogs and cats and may be
eective as an aid in the management of select clinical disorders.
Further data is needed to detail the range of immune mod-
ulation and to provide comparative data among probiotics.
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