Effect of the 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.

Introduction

Research on the use of probiotics, defined as living microorganisms 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 different 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 activity 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 immunoglobulin 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 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 immunosuppression 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 examination, 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 sensitivity 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 ethical 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 nutritional 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 × 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 vomiting, 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 concentrations 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.

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).

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

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).

Table II.

Hematologic values for the dogs before and after treatment

	Treatment; time of measurement; group mean ± standard deviationa
	Probiotic		Placebo
Variable	Before	After	Before	After
Hematocrit (%)	37.88 ± 7.65	40.23 ± 7.92	39.50 ± 9.61	42.26 ± 8.55
Hemoglobin concentration (g/dL)	11.89 ± 2.95	12.76 ± 2.13	12.63 ± 3.27	13.06 ± 2.88
Total plasma protein concentration (g/dL)	6.76 ± 3.04	6.95 ± 2.86	5.89 ±1.58	6.26 ± 1.58
Albumin concentration (g/dL)	4.38 ± 1.86	4.60 ± 1.90	3.45 ± 1.23	3.41 ± 0.90
Leukocyte count (per mm3)	9505 ± 4313	9732 ± 3622	21 997 ± 14 202	16 385 ± 8199

^aFor all the variables the difference between the mean values obtained before and after treatment was not significant (P > 0.05).

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 necessary 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 probiotics 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 influences 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 influence 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 growing dogs a dry food supplemented with a live probiotic enhanced long-term immune functions, including increased induction of specific 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 diarrhea and the time of hospitalization in comparison with the control patients (22).

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.