Effect of probiotic Lactobacillus murinus LbP2 on clinical parameters of dogs with distemper-associated diarrhea

Abstract

The objective of this study was to assess the effect of the probiotic Lactobacillus murinus native strain (LbP2) on general clinical parameters of dogs with distemper-associated diarrhea. Two groups of dogs over 60 d of age with distemper and diarrhea were used in the study, which was done at the Animal Hospital of the Veterinary Faculty of the University of Uruguay, Montevideo, Uruguay. The dogs were treated orally each day for 5 d with the probiotic or with a placebo (vehicle without bacteria). Clinical parameters were assessed and scored according to a system specially designed for this study. Blood parameters were also measured. Administration of the probiotic significantly improved the clinical score of the patients, whereas administration of the placebo did not. Stool output, fecal consistency, mental status, and appetite all improved in the probiotic-treated dogs. These results support previous findings of beneficial effects with the probiotic L. murinus LbP2 in dogs. Thus, combined with other therapeutic measures, probiotic treatment appears to be promising for the management of canine distemper-associated diarrhea.

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118 The Canadian Journal of Veterinary Research 2017;81:118–121
Introduction
Research on the use of probiotics, defined as living microorgan-
isms that when administered in adequate amounts confer a health
benefit on the host, may lead to promising results for the control of
infectious diseases in animals (1,2). These microorganisms have been
successfully used for the treatment of diarrhea associated with differ-
ent causes, such as antibiotic treatment, viral or bacterial infection,
travel (“traveller’s diarrhea”), and chemotherapy (3). Most of these
organisms have been obtained from feces or from the gut mucosa
of healthy individuals (1). This approach is supported by the results
of several studies proposing that native probiotic strains would be
well-adapted to the target ecologic niche and could successfully
compete in that environment (4). Probiotics can influence the gut
environment and the epithelial barriers in different ways since they
exert effects on junction stability between epithelial cells, mucus
production, mucosal immune response, and even commensal or
pathogenic microorganisms (5).
Lactobacillus murinus is one of the dominant lactobacilli in the
intestinal microbiota of the domestic dog (Canis lupus familiaris)
(6). In the present study an L. murinus strain (LbP2) was used for
the treatment of distemper-associated diarrhea in dogs. In previous
studies in our laboratory this strain had shown antimicrobial activ-
ity against Escherichia coli and Clostridium perfrigens (7). In addition,
enteric persistence of LbP2 had been observed in an in-vivo trial in
dogs in which it was also observed that the composition of the dogs’
intestinal microbiota as well as blood and body parameters were
not affected (8). As well, L. murinus LbP2 significantly influenced
the immune response: the production of total canine fecal immuno-
globulin A (IgA) significantly increased after oral administration (9).
This could reflect the ability of the probiotic to modulate the immune
response at a mucosal level, a potential key factor in the resolution
of diarrhea with various causes (10).
Canine distemper, which is distributed worldwide, is caused by
a morbillivirus of the family Paramyxoviridae and was originally
described in the 19th century. The host spectrum of this virus
Effect of the probiotic Lactobacillus murinus LbP2 on clinical parameters
of dogs with distemper-associated diarrhea
Luis Delucchi, Martín Fraga, Pablo Zunino
Abstract
The objective of this study was to assess the effect of the probiotic Lactobacillus murinus native strain (LbP2) on general clinical
parameters of dogs with distemper-associated diarrhea. Two groups of dogs over 60 d of age with distemper and diarrhea were
used in the study, which was done at the Animal Hospital of the Veterinary Faculty of the University of Uruguay, Montevideo,
Uruguay. The dogs were treated orally each day for 5 d with the probiotic or with a placebo (vehicle without bacteria). Clinical
parameters were assessed and scored according to a system specially designed for this study. Blood parameters were also
measured. Administration of the probiotic significantly improved the clinical score of the patients, whereas administration of
the placebo did not. Stool output, fecal consistency, mental status, and appetite all improved in the probiotic-treated dogs. These
results support previous findings of beneficial effects with the probiotic L. murinus LbP2 in dogs. Thus, combined with other
therapeutic measures, probiotic treatment appears to be promising for the management of canine distemper-associated diarrhea.
Résumé
L’objectif de la présente étude était d’évaluer l’effet d’une souche indigène du probiotique Lactobacillus murinus (LbP2) sur des paramètres
cliniques généraux de chiens avec une diarrhée associée au distemper. Deux groupes de chiens âgés de plus de 60 jours avec du distemper et
de la diarrhée ont fait partie de l’étude réalisée à l’hôpital de la Faculté vétérinaire de l’Université de l’Uruguay, Montevideo, Uruguay. Les
chiens ont été traités quotidiennement par voie orale pendant 5 j avec le probiotique ou avec un placebo (véhicule sans les bactéries). Des
paramètres cliniques ont été mesurés et notés selon un système conçu spécifiquement pour l’étude. Des paramètres sanguins ont également été
mesurés. L’administration du probiotique améliorait significativement le pointage clinique des patients, alors que l’administration du placebo
n’avait aucun effet. La production de selles, la consistance fécale, l’état mental, et l’appétit se sont tous améliorés chez les chiens traités avec
le probiotique. Ces résultats confirment des résultats antérieurs d’effets bénéfiques du probiotique L. murinus LbP2 chez les chiens. Ainsi,
combiner à d’autres mesures thérapeutiques, le traitement probiotique semble prometteur pour la gestion de diarrhée canine associée au distemper.
(Traduit par Docteur Serge Messier)
Departamento de Patología y Clínica de Pequeños Animales, Facultad de Veterinaria, Universidad de la República, Lasplaces 1550, CP 11600,
Montevideo, Uruguay (Delucchi); Departamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Avenida Italia 3318,
CP 11600, Montevideo, Uruguay (Fraga, Zunino).
Address all correspondence to Dr. Pablo Zunino; telephone: 598-24871616; fax: 598-24875461; e-mail: pzunino@iibce.edu.uy
Dr. Fraga’s current address is Plataforma de Investigación en Salud Animal, INIA La Estanzuela, Ruta 50 Km 11, Colonia, Uruguay.
Received May 6, 2016. Accepted September 22, 2016.
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2000;64:0–00 The Canadian Journal of Veterinary Research 119
includes numerous families of the Carnivora order, including Canidae
(dogs, foxes, and dingoes), Procyonidae (raccoons), Mustelidae (ferrets
and mink), and Felidae (large felids) (11). At the beginning of the
disease, dogs are depressed, anorexic, and febrile. The infection then
provokes a variety of clinical problems, affecting the gastrointestinal
and respiratory tracts and the skin and causing immunosuppres-
sion and demyelinating leukoencephalitis (11), as well as catarrhal
enteritis with depletion of the Peyer’s patches (12).
The aim of the present work was to assess the effect of orally
administered L. murinus LbP2 on clinical parameters of dogs with
distemper-associated diarrhea.
Materials and methods
Animals
Nineteen dogs with distemper treated at the Animal Hospital
of the Veterinary Faculty, University of Uruguay, Montevideo,
Uruguay, were included in the study. Besides clinical examina-
tion, the disease was diagnosed by means of a Canine distemper
virus (CDV) test kit involving the use of nasal or ocular discharges
and a sandwich lateral-flow immunochromatograph (Isu Abxis
Company, Seoul, Korea). According to the manufacturer, the sensi-
tivity and the specificity of the test are 100% and 98%, respectively.
The samples were taken with a swab and diluted in the buffer
provided by the manufacturer. Then 3 drops were placed in the
cassette for the diagnostic procedure. All the dogs included in
the study showed distemper-positive results according to the test
interpretation.
All procedures were approved by the Veterinary Faculty’s
Bioethics Committee and carried out under the current national ethi-
cal regulations (Law 18.611, approved by the Uruguayan Parliament
March 28, 2014). The dogs were more than 60 d old (the oldest was
aged 8 y) and had diarrhea and other digestive signs. The exclusion
criteria were any of poor clinical status, severe weakness, bad nutri-
tional status, and depressed mental status, as well as treatment with
antibiotics, probiotics, steroids, or other drugs in the previous 72 h.
Nutritional support during the days of the study consisted of
ad-libitum availability of commercial food (Hill’s Prescription Diet
a/d Canine/Feline; Hill’s Pet Nutrition, Topeka, Kansas, USA).
According to the dog’s clinical condition, Ringer’s solution and
metoclopramide (1 mg/kg per day) were supplied intravenously
to the dogs that vomited.
The study was designed as a randomized controlled trial; 13 dogs
were to be treated with L. murinus LbP2 and the other 7 dogs with
a placebo.
Probiotic and placebo preparation and
administration
Lactobacillus murinus LbP2 was grown on MRS agar from stock
cultures that had been stored at −80°C (Becton, Dickinson and
Company, Franklin Lakes, New Jersey, USA); some colonies were
picked and cultured in MRS broth. Both incubations were done for
24 h under microaerophilic conditions at 37°C. After 3 washes in
sterile phosphate-buffered saline, bacteria were inoculated in 10%
autoclaved skim milk (Becton, Dickinson and Company) for a final
bacterial dose of 5 3 109 colony-forming units and lyophilized with
a Freezone Stoppering Tray Dryer (Labconco, Kansas City, Missouri,
USA). The vials were stored at 4°C, and contamination and viability
controls were done at weeks 3 and 6. The placebo consisted of 10%
autoclaved skim milk only, which was lyophilized and stored under
the same conditions as the probiotic. Probiotic and placebo were
rehydrated with 5 mL of sterile water and administered orally for
5 d, a single dose per day.
Clinical assessment
A clinical score based on 5 items was designed. It included the
objective signs of stool output and consistency, along with vomit-
ing, and the subjective signs of appetite and mental status (alert
or depressed) (13). The normal value for each item was 1; 0 was
assigned when the condition was altered. Normal stool output was
considered to be fewer than 3 deposits per day. Stool consistency
was scored as 0 (watery) to 5 (solid). These 5 items were chosen
because of their frequency among clinical signs at presentation
in the digestive form of the disease according to a retrospective
study of canine distemper cases at the Veterinary Hospital over
13 y (14).
Blood samples were taken from the cephalic vein on days 0 and 6
and analyzed at the Clinical Laboratory of the Veterinary Faculty for
hematocrit, hemoglobin level, total leukocyte count, and concentra-
tions of total plasma proteins and albumin.
Data analysis
The Wilcoxon test was used to compare the clinical scores within
the 2 groups of dogs, and the Mann–Whitney U-test was used to
compare the scores between the 2 groups. Student’s t-test was used
to compare the hematologic parameters between the 2 groups.
Differences were considered significant when P-values were , 0.05.
Table I. Clinical scores for dogs with distemper-associated
diarrhea treated with the probiotic Lactobacillus murinus
LbP2 or with placebo
Treatment (and number of dogs);
time of assessment; group score and
P-value for the difference in score
between those times
Probiotic (12) Placebo (7)
Variable Before After Before After
Mental status 3 5 1 1
0.0253 1
Appetite 1 5 0 1
0.0455 0.3173
Stool consistency 1 5 0 1
0.0455 0.3173
Stool output 4 7 1 2
0.0832 0.3173
Vomiting 7 8 4 2
0.6547 0.1573
Total 16 30 6 7
0.0277 0.7054
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120 The Canadian Journal of Veterinary Research 2000;64:0–00
Results
In the group to be treated with the probiotic, 1 dog died 72 h
before the start of treatment. Of the remaining 12 dogs 7 improved
according to the general clinical score and 1 dog worsened; in the
other 3 dogs the score did not change. Score variation over time was
statistically significant (P = 0. 0277) (Table I). Of the 7 dogs treated
with the placebo, 2 improved slightly according to the clinical score
and 2 worsened; in the other 3 dogs the values did not change. No
significant differences were observed between the scores before and
after treatment in the placebo group (P = 0.70546) (Table I).
Significant differences were observed between the clinical scores
of the 2 groups (P = 0.0357). The score items that influenced the
final P-value were stool consistency (P = 0.0455), mental status
(P = 0.0253), and appetite (P = 0.0455). Although the effect of
treatment on stool output was not significant, a clear tendency
(P = 0.0832) could be seen.
No significant differences in hematologic parameters between
initial and final values were observed in either group during the
course of treatment (Table II).
Discussion
In recent years the use of probiotics has become a promising tool
for the treatment and prevention of different diseases, including
infections of varied cause, and their use in companion animals has
progressively increased (15). Probiotic properties are strain-specific
and vary within the same bacterial species (16). Therefore, it is neces-
sary to thoroughly characterize the properties of the different strains
that are potentially associated with therapeutic value.
In this study an L. murinus strain extensively characterized by our
group was used as a therapeutic support for the treatment of dogs
with distemper-associated diarrhea. It had already been reported that
different L. murinus strains showed interesting properties as probiot-
ics with in-vitro approaches (7,17). Although the use of probiotics has
consistently increased in recent years in both humans and animals,
there have been few studies evaluating the use of probiotics in cases
of canine diarrhea. Herstadet et al (18) conducted a controlled clinical
trial that included 36 dogs with diarrhea treated with a commercial
probiotic based on Lactobacillus spp., Pediococcus spp., and Bacillus
spp. of different origins. The authors observed that the time between
the start of the treatment and the last abnormal fecal deposit was
significantly reduced in the treated group compared with the group
that received the placebo.
In our study L. murinus LbP2 showed a beneficial effect on the
clinical condition of dogs with distemper, probably exerting its
effects on the intestinal barrier and immunity. In a previous study
we proved that this strain significantly induced the production
of enteric IgA (9). This effect shows the ability of the probiotic to
modulate the immune response, so it is possible that it also influ-
ences enteric inflammation, improving absorption of nutrients and
contributing to appetite stimulation and food intake. In this study
appetite stimulation was seen in the dogs treated with the probiotic
but not in those treated with the placebo.
In a previous work no significant differences were found among
the main groups of enteric bacteria of dogs treated with L. murinus
LbP2 or with placebo (8). Although further studies are required, we
could consider that the probiotic effects are not related to an influ-
ence of the principal groups in the gut microbiota.
In this study the hematologic parameters of the 2 groups of dogs
did not show any significant difference related to treatment. This
can be taken as an advantage in the use of L. murinus LbP2 since
alterations in hematologic parameters, especially the blood leukocyte
profile, could be an indicator of risk associated with its use. Clinical
use and safety of these live microorganisms in humans and animals
are under debate (19). Our results confirm previous findings that
L. murinus LbP2 did not show any deleterious effects in canines (8).
It is also important to consider that L. murinus LbP2 maintained
its therapeutic value as a probiotic even after lyophilization since
in some cases this preservation procedure can affect a probiotic’s
effects (20).
As far as we know, there are no published reports on the use of
probiotics for the management of diarrhea associated with canine
viruses. However, Benyacoub et al (21) reported that feeding grow-
ing dogs a dry food supplemented with a live probiotic enhanced
long-term immune functions, including increased induction of spe-
cific antibodies by a live-attenuated vaccine against CDV. Studies
that included the use of probiotics for the treatment of children with
rotavirus-associated diarrhea and acute diarrhea found a reduced
number of days of diarrhea with probiotic supplementation (21).
Specifically, in children affected by rotavirus, the use of Saccharomyces
boulardii reduced the time to the resolution of vomiting and diar-
rhea and the time of hospitalization in comparison with the control
patients (22).
Table II. Hematologic values for the dogs before and after treatment
Treatment; time of measurement; group mean 6 standard deviationa
Probiotic Placebo
Variable Before After Before After
Hematocrit (%) 37.88 6 7.65 40.23 6 7.92 39.50 6 9.61 42.26 6 8.55
Hemoglobin concentration (g/dL) 11.89 6 2.95 12.76 6 2.13 12.63 6 3.27 13.06 6 2.88
Total plasma protein concentration (g/dL) 6.76 6 3.04 6.95 6 2.86 5.89 61.58 6.26 6 1.58
Albumin concentration (g/dL) 4.38 6 1.86 4.60 6 1.90 3.45 6 1.23 3.41 6 0.90
Leukocyte count (per mm3) 9505 6 4313 9732 6 3622 21 997 6 14 202 16 385 6 8199
a For all the variables the difference between the mean values obtained before and after treatment was not significant
(P . 0.05).
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2000;64:0–00 The Canadian Journal of Veterinary Research 121
In canines, the treatment of secretory diarrhea is a therapeutic
challenge because there is no etiologic treatment in most cases,
and when there is specific treatment it is lengthy, prolonging the
animal’s state of weakness. Viral diseases such as canine distemper
occur with these types of signs. Also, as diarrhea is a common sign
of many diseases, the indiscriminate use of antibiotics can favor the
emergence of resistant microorganisms, aggravating the clinical
picture by removing the native microbiota or causing overgrowth
of pathogenic bacteria. Considering all these factors and taking into
account the results of the present study, the use of probiotics appears
to be a promising tool for the management of diarrhea, particularly
diarrhea associated with canine distemper.
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