Scholarly article on topic 'Experimental addition of Eleutherococcus senticosus and probiotic to the canine diet'

Experimental addition of Eleutherococcus senticosus and probiotic to the canine diet Academic research paper on "Veterinary science"

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Academic research paper on topic "Experimental addition of Eleutherococcus senticosus and probiotic to the canine diet"

VERSITA

Central European Journal of Biology

Experimental addition of Eleutherococcus senticosus and probiotic to the canine diet

Research Article

Viola Strompfovä1*, Iveta Plachä1, Klaudia Cobanova1, Sona Gancarcikova2,

Dagmar Mudronova2, Andrea Laukova1

Institute of Animal Physiology, Slovak Academy of Sciences,

040 01 Kosice, Slovakia

2University of Veterinary Medicine and Pharmacy, Department of Microbiology and Immunology,

041 81 Kosice, Slovakia

Received 21 October 2011; Accepted 07 February 2012

Abstract: There is a current trend to support pet health through the addition of natural supplements to their diet, taking into account the high incidence of medical conditions related to their immune system and gastrointestinal tract. This study investigates effects of the plant Eleutherococcus senticosus as a dietary additive on faecal microbiota, faecal characteristics, blood serum biochemistry and selected parameters of cellular immunity in healthy dogs. A combination of the plant with the canine-derived probiotic strain Lactobacillus fermentum CCM 7421 was also evaluated. Thirty-two dogs were devided into 4 treatment groups; receiving no additive (control), dry root extract of E. senticosus (8 mg/kg of body weight), probiotic strain (108 CFU/mL, 0.1 mL/kg bw) and the combination of both additives. The trial lasted 49 days with 14 days supplementation period. Results confirm no antimicrobial effect of the plant on the probiotic abundance either in vitro (cultivation test) or in vivo. The numbers of clostridia, lactic acid bacteria and Gram-negative bacteria as well as the concentration of serum total protein, triglyceride, glucose and aspartate aminotransferase were significantly altered according to the treatment group. Leukocyte phagocytosis was significantly stimulated by the addition of probiotic while application of plant alone led to a significant decrease.

Keywords: Probiotics • Lactobacillus fermentum • Eleutherococcus senticosus • Gut microflora • Serum biochemistry • Cellular immunity © Versita Sp. z o.o.

1. Introduction

The incidence of special health problems in dogs has shown a rapid increase in recent years. Veterinary Pet Insurance analysis reported ear infections, skin allergies and infections, gastritis and enteritis as the top five canine medical conditions of 2010 [1]. Many factors may be involved (medical drugs and vaccines, environmental pollutants, lack of exercise, excess stress, genetic factors etc.). However, inappropriate nutrition could probably be considered as the critical cause since the gastrointestinal (GI) system plays a central role in immune system homeostasis necessary to prevent the mentioned diseases. The importance of immune modulation at the GI level can be understood easily, considering that approximately 70% of the entire immune system is found at this site and that in the

lamina propria there are about 80% of all plasma cells responsible for IgA antibody production [2,3]. Neverless, the ageing process impaires cell-mediated immunity as was observed also in dogs [4].

The use of bioactive plant species as a dietary additive alone or combined with probiotic microorganisms could be helpful in the prevention of impaired immune functions, although scientific knowledge on their effects in canine organism is scarce [5-7]. Despite that, most recent surveys show an upward trend in using phytoproducts by veterinarians (e.g. three-quarters of the veterinarians in Austria, Germany and Switzerland, [8]).

The medicinal herb Eleutherococcus senticosus (Rupr.& Maxim.) Maxim. (Araliaceae) is an approximately two-meter high, thorny shrub native to the far eastern areas (China, Korea, Japan, Russian Far East) where it is commonly referred to as ciwujia. This plant, known

E-mail: strompfv@saske.sk

£ Springer

as an adaptogen, has been shown to have a wide range of other pharmacological effects as observed in studies in humans or mice: antistress, antioxidant, antitumor, immunostimulatory, antiinflammatory, antipyretic, hypocholesterolemic, hypoglycemic, choleretic and radioprotectant activity [9-11]. The active compounds isolated from E. senticosus include phenylpropanoids (e.g syringin, caffeic acid, sinapyl alcohol, coniferyl aldehyde), lignans (e.g. sesamin, syringoresinol and its glucoside), saponins (e.g. daucosterol, b-sitosterol, hederasaponin B), coumarins (e.g. isofraxidin and its glucoside), vitamins (vitamin E) and provitamins (b-carotene, [9]).

The current trend of feeding dogs by commercial food products with reduced microbial populations (heated above 100°C) results in an insufficient microbial exposure which is required for the induction of immune mechanisms. Probiotic supplementation of canine diet may provide a promising alternative to promote an effective gut defense barrier. Probiotic microorganisms display a wide range of positive effects although strain specific, but overall include a trophic action on the intestinal mucosa (SCFA, vitamin and enzymes production), competitive exclusion of enteric pathogens, production of pathogen inhibitory substances (lactic acid, bacteriocin, etc.), inhibition of microbial toxin action, neutralization of dietary carcinogens, antioxidant activity and immunomodulation [12,13]. Some probiotic strains reduce pathological alterations in paracellular permeability to large molecules or bacteria, degrade and modify the structure of antigen macromolecules, reduce mucus degradation, stimulate mucosal immunity and modulate the production of inflammatory mediators in the intestinal epithelium [14-16].

Since no investigation has been conducted on the influence of E. senticosus on the overall health status of dogs, we decided to test the effects of E. senticosus extract by oral application alone (ES group) and in combination with canine-derived probiotic strain L. fermentum CCM 7421 (ES+LF group) on the faecal microbiota, faecal characteristics, serum biochemistry and cellular immunity in healthy dogs. The study included the L. fermentum CCM 7421 animal group receving the probiotic strain alone (LF group).

2. Experimental Procedures

2.1. In vitro assay

The effect of E. senticosus extract on the growth of probiotic L. fermentum CCM 7421 strain was tested in vitro in order to be sure that the extract and the strain can be combined in the in vivo experiment. Dry

root extract of Eleutherococcus senticosus (ethanolic extraction, dry matter 95±1%, ash 7.3±0.5%, purchased from Calendula, a.s., Nova Lubovna, Slovakia) was added to de Man-Rogosa-Sharpe broth (MRS, pH 5.58, Merck, Germany) in the amount of 1.0, 5.0, 10.0 or 20.0 g/L of broth. Each broth was inoculated (1% w/v) with an 18 h preculture and incubated at 37°C for 24 h aerobically. The pH and growth - CFU/mL (colony forming units) was determined on MRS agar plates (Merck) at time 0 and after 24 h of cultivation. The MRS broth inoculated with CCM 7421 strain and without extract addition served as the control.

2.2 Animals and diet

Healthy adult dogs (n=32; 18 females, 14 males) were randomly divided into four experimental groups, 8 animals in each. The age of the dogs ranging between 1-11 years (mean age 2.7±1.1) and body weights between 19.2 and 44.0 kg (mean BW 31.0±7.9 kg). They belong to the following breeds: German Shepherd n=20, Belgian Shepherd (Malinois) n=3, cross-breed n=3, Rottweiler n=2, Doberman n=2, German Shorthaired Pointer n=1, Rhodesian Ridgeback n=1. All experimental procedures were approved by the Ethic Commission of the Institute of Animal Physiology, Slovak Academy of Sciences (Kosice, Slovakia). Dogs were housed individually in a whole environment, but covered, facility measuring 3.0x3.0 m with box 1.5x0.8 m (temperature, 10-15°C). They were fed and exercised individually and had access to fresh water at all times. They received a commercial, nutritionally complete, extruded dry dog food twice a day (Purina Pro Plan Dog Adult Medium Breed, Chicken & Rice formula, Netsle Purina PetCare Company, St. Louis, USA), which cointained (g/100g diet): crude protein 27.0, crude fat 17.0, crude fibre 2.0, ash 7.0, calcium 1.3, phosphorus 1.0 (sodium selenite 0.015 mg, copper sulphate 2 mg, vitamin A 2100 IU, vitamin E 17 IU, vitamin D3 160 IU, ascorbic acid 7 mg), 3580 kcal metabolizable energy/kg. Adaption period to this food was 4 weeks before experiment.

The experiment lasting 49 days was composed of a baseline (day 0), supplementation (day 1-14) and post-supplementation period (day 15-49). The animals were assigned to the following experimental groups: the control (C, n=8) without any treatment, the ES group (n=8) supplemented with dry root extract of Eleutherococcus senticosus (at a dose 8 mg/kg body weight), the LF group (n=8) supplemented with L. fermentum CCM 7421 (0.1 mL/kg BW, 108 CFU/mL of Ringer buffer, Merck, Germany), and the ES+LF group (n=8) fed the combination of root powder and a probiotic culture at the same doses as the previous groups. Dogs were supplemented with the appropriate additive daily

during the feeding time (for 14 days) and monitored for changes in clinical condition, vital parameters and appetite throughout the study.

2.3 Preparation of L. fermentum CCM 7421 for application to dogs

The probiotic characteristics of strain L. fermentum CCM 7421 (AD1, isolate from canine faeces) have been previously presented [7]. A rifampicin-marked strain of L. fermentum CCM 7421 was prepared by subsequent cultivation on MRS agar plates (Merck) supplemented with 100 |jg/mL of rifampicin. The strain was then cultivated in MRS broth (Merck) at 37°C for 24 h. Cells were harvested after centrifugation (10 min at 2000xg) and the culture sediment was resuspended in Ringer buffer (Merck, pH 7.0) to a concentration of 108 CFU/mL. The solution was stable for 1 week at 4°C. Then it was replaced by a new cell culture.

2.4 Sampling procedures

Fresh faecal samples were collected at days 0 (pre-treatment), 7, 14 (treatment), 21, 28, and 49 (post-treatment period) during morning individual walking to ensure that faeces were correctly allocated to the proper animal. The determination of faecal score and pH measurement were perfomed immediately. Blood samples (from vena cephalica antebrachii) were collected at days 0, 14, 28, and 49 in plastic tubes containing (1) 10 UI heparin (20 pl/mL of blood) for phagocytic activity, glutathione peroxidase determination, (2) 1.5% EDTA (0.1 mL/mL of blood) for iodo-nitro-tetrazolium reductase test, and (3) without anticoagulant for determination of biochemical parameters. The dogs were not allowed access to food in the 16-hour overnight period prior to venipuncture. The blood samples for testing of biochemical parameters were centrifuged (15 min at 2500xg) after 30 min and sera frozen at -20°C.

2.5 Microbiological analysis

The samples of faeces (1 g) were mixed with sterile Ringer buffer (Merck, pH 7.0) and homogenized (3 min) using a stomacher (IUL, Instruments, Spain). Microbial populations were determined according to the standard microbiological method by serial dilution (10-1 to 10-7). Aliquots of the dilutions (100 jL) were inoculated onto the following selective media: Mac Conkey agar for enumeration of E. coli (Becton and Dickinson, USA), Mannitol salt agar for staphylococci (Oxoid, UK), M-Enterococcus agar for enterococci (Becton and Dickinson), MRS agar for lactic acid bacteria (LAB, Becton and Dickinson), MRS agar with rifampicin (100 jg/mL) for L. fermentum CCM 7421, Pseudomonas agar (Biomark, India) for Pseudomonas sp.,

Aeromonas medium base (Oxoid) for Aeromonas sp., and Clostridium difficile agar base (Oxoid) for clostridia. Plates were incubated aerobically at 37°C for 24-48 h except for Aeromonas and Pseudomonas plates incubated at 25°C for 48-72 h. Clostridia were cultivated anaerobically (Bactron Anaerobic Chamber, Shel Lab, Sheldon Manufacturing Inc., USA, athmosphere composition 90% N2 + 5% H2 + 5% CO2) at 38°C for 48 h. The results were expressed as log10 CFU per gram of moist faeces.

2.6 Faecal characteristics

Fresh faecal samples were visually scored (FS-faecal score) according to the following system: 1 = hard dry faeces; 2 = hard, formed stool; 3 = soft, formed, and moist stool; 4 = soft, unformed stool; 5 = watery liquid. Detection of pH (pH Meter, Hanna Instruments, USA) was performed immediatelly. Approximately 5 g of samples were stored at -20°C until analysis of dry matter (DM). Ammonia concentration was tested using an Ammonia Assay Kit (Sigma-Aldrich, USA).

2.7 Blood analysis

Biochemical parameters in blood serum were determined by colorimetric methods (Spectrophotometer UV-2550 Shimadzu, Japan) using kits (Randox Laboratories Ltd., UK) for the following parameters: total protein (TP 245), albumin (AB 362), urea (UR107), triglyceride (TR 210), cholesterol (CH 200), glucose (GL 2623), alanine aminotransferase (AL 100), aspartate aminotransferase (AS 147), calcium (CA590), inorganic phosphorus (PH 1016), glutathione peroxidase (RS 504). Phagocytic activity (PA) was tested microscopically after Pappenheim staining according the method of Steruska [17] and expressed as percentage of phagocyte ingested yeast cells to the total number of phagocytes (100 PMNs were counted per sample). Measurement of tetrazolium reductase activity of phagocytes was performed according to the technique described by Lokaj and Oburkova [18] and expressed as index of metabolic activity (IMA) based on the ratio of mean optical density (OD485) of leukocyte suspension with starch (stimulated phagocytic cells) to the leukocyte suspension without the starch (negative control).

2.8 Statistical analysis

The results are expressed as the mean + standard error. Statistical analyses were performed with GraphPad Prism software (version 5.0). Data were analyzed for the the effects of treatment, time and treatment x time interaction using two-way analysis of variance (ANOVA, Bonferroni post-test) with the level of significance set at P<0.05.

3. Results

3.1 In vitro assay

The presence of E. senticosus extract in MRS broth in the concentrations of 0.1, 0.5, 1.0 and 2.0% only marginally affects the growth of probiotic L. fermentum CCM 7421 strain. We observed an inhibition by just 0.4 log10 CFU/mL at 2.0% concentration compared to the control (Table 1). After 24 h cultivation, no significant differences in the pH values of cultures and control were observed. In addition, the initial pH value of MRS broth was slightly decreased with increasing amount of plant extract added (acidifing effect of extract).

group significant reduction occurred only in the post-treatment phase (days 21 and 28). Numbers of faecal enterococci were not affected by the time or treatment (P>0.05). The population of E. coli was more abundant in the ES+LF group compared to groups with application of additives only (ES and LF group, P<0.05) during the supplementation phase (day 7 and 14). Numbers of other tested gram-negative genera (Pseudomonas sp., Aeromonas sp.) were similarly higher in the combined ES+LF group when compared to the ES group. The difference between these groups was significant at day 14 (P<0.01). An effect of time in the Pseudomonas population was also noted (P=0.0065).

3.2 Microbial populations in faeces

In the canine experiment, oral supplementation with E. senticosus extract (ES group), L. fermentum CCM 7421 (LF group) and their combination (ES+LF group) resulted in certain significant effects according to the microbial genera tested in faecal samples (Table 2). The population of LAB was higher in both groups supplemented with the probiotic (LF and ES+LF) compared to the ES group with a significant difference detected at day 28 and 49 (P<0.01 and P<0.05, respectively). In addition, a nonsignificant decrease of LAB numbers in the LF and ES+LF group shortly after the cessation of the probiotic supplementation (day 21) was replaced again by an increase in the later post-treatment phase (day 28 and 49). The probiotic strain CCM 7421 was detected in faecal levels up to 106 CFU/g on average (day 7), but later the numbers decreased to 103-102 CFU/g (day 28 and 49). Thus a significant effect of time was observed (P=0.0045). The treatment effect (P=0.0044) was observed also in clostridia numbers which were significantly lower in the LF and ES+LF group in the early post-treatment period (day 21, P<0.05, compared to the ES). However, the lowering effect in the LF started already at the end of supplementation phase (day 14). In contrast, the ES+LF

3.3 Faecal characteristics

The application of L. fermentum CCM 7421 caused a significant decrease of faecal pH value in the dogs of LF group (Table 3). The decrease was significant compared to other treated groups, especially at day 7, 14 and 28 (P<0.01). In contrast, pH values in the ES and ES+LF groups tended to be higher than the control levels (P>0.05) between day 7 and 28. No treatment or time effect was noted (P>0.05) with the consistency of faeces. However, faeces from both groups fed the E. senticosus extract (ES, ES+LF) showed lower DM content than the LF group (day 49, P<0.05) and more liquid consistency also in FS in the ES+LF group (day 14, P<0.05). An effect of treatment (P<0.05) on the faecal ammonia concentrations was observed whereby samples of dogs in the ES+LF group showed lower values compared to that of the LF group (day 14, P<0.05; day 49, P<0.05). This difference in the DM base was not significant (P>0.05).

3.4 Cellular immunity assay and biochemistry in blood

The singular application of E. senticosus to dogs resulted in a significant decrease of leukocyte

Concentration Growth (log10 CFU/mL) Time (h) pH of culture

(% w/v) 0 24 0 24

0.0 (control) 6.27 ± 0.12 8.09 ± 0.19 5.49 ± 0.05 3. 9 3 ± 0.03

0.1 6.12 ± 0.30 8.26 ± 0.24 5.48 ± 0.09 3.9 3 ± 0.05

0.5 6.46 ± 0.10 8.13 ± 0.12 5.46 ± 0.05 3.9 3 ± 0.08

1.0 6.81 ± 0.04 8.56 ± 0.07 5.41 ± 0.10 3. 9 3 ± 0.03

2.0 6.38 ± 0.08 7.81 ± 0.02 5.35 ± 0.07 3.9 3 ± 0.02

Table 1. In vitro growth of L. fermentum CCM 7421 in MRS broth (Merck, pH 5.58) without and with the addition of E. senticosus dry root extract at a concentrations of 0.1, 0.5, 1.0 and 2.0% (w/v).

Values are means of two replicates ± SEM

Time (d) P Value

Microorganism Exper. group 0 7 14 21 28 49 Effect of treatment Effect of time Inter-actlon

C 5.60 ± 0.30 5.66 ± 0.24 5.68 ± 0.35 5.66 ± 0.32 5.67 ± 0.21 5.71 ± 0.14

Aeromonas sp. ES LF 5.59 ± 0.27 5.53 ± 0.43 5.35 ± 0.29 5.03 ± 0.54 4.34 ± 0.48A 5.08 ± 0.26 5.35 ± 0.64 5.39 ± 0.34 4.60 ± 0.36 4.88 ± 0.35 5.47 ± 0.65 5.71 ± 0.64 0.0001 0.6286 0.8782

ES + LF 5.62 ± 0.53 6.24 ± 0.41 6.46 ± 0.75B 6.32 ± 0.49 6.17 ± 0.22 6.37 ± 0.45

C 5.55 ± 0.21 5.50 ± 0.18 5.50 ± 0.32 5.56 ± 0.27 5.59 ± 0.25 5.60 ± 0.19

Clostridlum-like ES LF 5.60 ± 0.26 5.62 ± 0.29 5.88 ± 0.19 5.13 ± 0.29 5.46 ± 0.45 3.85 ± 0.57 6.23 ± 0.21a 4.33 ± 0.42b 5.10 ± 0.75 4.42 ± 0.50 5.04 ± 0.97 5.32 ± 0.77 0.0044 0.1948 0.4636

ES + LF 5.51 ± 0.68 5.15 ± 0.68 5.19 ± 0.73 4.25 ± 0.31b 3.76 ± 0.39 5.05 ± 0.76

C 4.67 ± 0.11 4.71 ± 0.20 4.65 ± 0.17 4.73 ± 0.20 4.68 ± 0.17 4.71 ± 0.22

Enterococcus sp. ES LF 4.86 ± 0.36 4.71 ± 0.34 3.86 ± 0.26 4.99 ± 0.26 4.43 ± 0.41 4.78 ± 0.34 4.83 ± 0.36 5.04 ± 0.36 4.34 ± 0.21 5.09 ± 0.40 5.50 ± 0.33 5.21 ± 0.42 0.2280 0.5894 0.8825

ES + LF 4.77 ± 0.75 5.17 ± 0.73 5.33 ± 0.84 5.41 ± 0.58 4.62 ± 0.39 5.08 ± 0.62

C 5.62 ±0.18 5.66 ± 0.21 5.69 ± 0.14 5.62 ± 0.12 5.65 ± 0.26 5.70 ± 0.26

Escherichia coli ES LF 5.90 ± 0.41 5.70 ± 0.32 5.72 ± 0.32 4.80 ± 0.42a 4.50 ± 0.56a 5.20 ± 0.25 5.52 ± 0.50 5.31 ± 0.33 5.16 ± 0.41 5.02 ± 0.42 5.98 ± 0.72 5.71 ± 0.55 0.0006 0.4399 0.7858

ES + LF 5.79 ± 0.68 6.78 ± 0.32b 6.27 ± 0.99b 5.97 ± 0.55 6.33 ± 0.40 6.83 ± 0.40

C 6.46 ± 0.23 6.57 ± 0.13 6.40 ± 0.27 6.50 ± 0.20 6.41 ± 0.18 6.51 ± 0.26

LAB ES LF 6.55 ± 0.47 6.49 ± 0.43 6.65 ± 0.37 7.86 ± 0.42 6.82 ± 0.13 6.72 ± 0.39 7.03 ± 0.26 6.55 ± 0.52 5.17 ± 0.83A 7.76 ± 0.68B 6.49 ± 0.43a 7.87 ± 0.26 <0.0001 0.1578 0.1391

ES + LF 6.44 ± 0.84 7.81 ± 0.81 8.03 ± 0.65 7.51 ± 0.58 7.63 ± 0.39B 8.28 ± 0.41b

L fermentum CCM 7421 LF ES + LF ND ND 5.73 ± 0.48 5.81 ± 0.90 4.69 ± 0.56 4.67 ± 1.30 4.00 ± 0.87 3.82 ± 0.47 2.95 ± 0.46 3.98 ± 0.63 2.17 ± 0.55 3.96 ± 0.79 0.2560 0.0045 0.6356

C 5.74 ± 0.28 5.75 ± 0.38 5.69 ± 0.21 5.73 ± 0.33 5.76 ± 0.21 5.80 ± 0.13A

Pseudomonas sp. ES LF 5.69 ± 0.36 5.93 ± 0.38 5.95 ± 0.27 6.45 ± 0.54 4.88 ± 0.33A 5.42 ± 0.38 5.78 ± 0.56 5.87 ± 0.45 5.20 ± 0.46 5.28 ± 0.52 6.19 ± 0.56 7.64 ± 0.36B 0.0020 0.0065 0.2604

ES + LF 5.86 ± 0.65 6.44 ± 0.52 6.83 ± 0.28B 6.27 ± 0.26 6.34 ± 0.21 6.89 ± 0.45

Table 2. Faecal microbial populations of control dogs (C, n=8), dogs fed E. senticosus (ES, n=8), L. fermentum CCM 7421 (LF, n=8) and the combination of both additives (ES+LF, n=8) for 14 days.

Values are means logm CFU/g ± SEM. Significant results: ab P<0.05; AB<0.01. ND - not detected.

Time (d) P Value

Parameter Exper. group 0 7 14 21 28 49 Effect Effect of treatment of time Interaction

C 6.3 ± 0.2 6.3 ± 0.1 6.3 ± 0.1 6.3 ± 0.2 6.3 ± 0.1 6.3 ± 0.1

ES 6.2 ± 0.1 7.0 ± 0.2A 6.4 ± 0.2 6.4 ± 0.2 6.8 ± 0.2A 6.2 ± 0.3

PH <0.0001 0.1347 0.1223

LF 6.3 ± 0.2 6.0 ± 0.2B 5.8 ± 0.1A 5.7 ± 0.1 5.8 ± 0.1B 6.2 ± 0.1

ES + LF 6.3 ± 0.3 7.0 ± 0.2A 6.9 ± 0.3B 6.4 ± 0.2 6.8 ± 0.4A 6.4 ± 0.3

C 3.2 ± 0.1 3.2 ± 0.2 3.2 ± 0.2a 3.1 ± 0.1 3.1 ± 0.1 3.2 ± 0.2

ES 3.2 ± 0.1 3.3 ± 0.2 3.1 ± 0.1A 3.3 ± 0.2 3.2 ± 0.2 3.2 ± 0.2

Faecal score 0.1448 0.7891 0.6374

LF 3.2 ± 0.1 3.3 ± 0.1 3.2 ± 0.1a 3.3 ± 0.1 3.3 ± 0.1 3.3 ± 0.1

ES + LF 3.1 ± 0.2 3.3 ± 0.2 3.9 ± 0.3bB 3.4 ± 0.2 3.3 ± 0.2 3.3 ± 0.2

C 29.8 ± 1.2 NT 30.0 ± 0.9 NT 30.1 ± 1.3 30.0 ± 3.2

ES 30.5 ± 1.2 NT 29.6 ± 2.3 NT 29.2 ± 1.5 24.7 ± 1.4a

Dry matter (%) 0.1965 0.4325 0.5865

LF 29.0 ± 0.8 NT 33.2 ± 1.7 NT 30.4 ± 1.2 31.1 ± 1.4b

ES + LF 29.6 ± 2.0 NT 29.0 ± 1.2 NT 29.1 ± 2.9 28.0 ± 1.4

C 0.71 ± 0.04 NT 0.73 ± 0.02 NT 0.74 ± 0.07 0.73 ± 0.03

ES 0.70 ± 0.04 NT 0.82 ± 0.08 NT 0.85 ± 0.11 0.64 ± 0.09

Ammonia (mg/g) 0.0016 0.5663 0.5875

LF 0.71 ± 0.04 NT 0.85 ± 0.04a NT 0.82 ± 0.06 0.86 ± 0.05a

ES+LF 0.68 ± 0.13 NT 0.57 ± 0.12b NT 0.61 ± 0.10 0.53 ± 0.09b

Table 3. Faecal characterstlcs of control dogs (C, n=8), dogs fed E. senticosus (ES, n=8), L. fermentum CCM 7421 (LF, n=8) and the combination of both additives (ES+LF, n=8) for 14 days. Values are means ± SEM. Significant results: ah P<0.05: AB P<0.01: NT - not tested.

phagocytic activity (PA) compared to the control, LF and ES+LF groups (P<0.01, day 28 and 49, Figure 1). On the contrary, both probiotic groups (LF, ES+LF) showed a significant increase of PA compared to the control at day 49 (P<0.01). The treatment effect (P<0.0001) and the interaction of treatment and time (P<0.0001) on the PA parameter and also in the index of phagocytic activity (IPA) was observed. Similarly, a decrease of IPA in the ES group (day 28 and 49, P<0.01 compared to all groups) and an increase, especially in the LF group, was detected (day 49, P<0.01 compared to the control and ES group). On the other hand, respiratory burst activity of leukocytes expressed as IMA was the highest in the dogs supplemented only the E. senticosus extract (ES group, day 14, P<0.05 compared to the control and ES+LF, Figure 2).

lime (<(>

Figure 1. Phagocytic activity of PMNs leukocytes in blood samples of the control (C, n=8), dogs fed E. senticosus (ES group, n=8), L. fermentum CCM 7421 (LF, n=8) and their combination (ES+LF group, n=8) for 14 days. Results are expressed as mean percentage ± SEM. Significant results: different letters P<0.01.

Figure 2. Respiratory burst activity of leukocytes In blood samples of the control (C, n=8), dogs supplemented with E. senticosus (ES group, n=8). L. fermentum CCM 7421 (LF group, n=8) and the combination of both additives (ES+LF, n=8) for 14 days. Results are expressed as index of metabolic activity (mean ± SEM). Significant result: different letters P<0.05.

According to our results, from the analysis of biochemical parameters in blood serum of dogs (Table 4), treatment effects on the concentration of total protein, triglyceride, glucose (with time effect) and aspartate aminotransferase were detected. Total protein was detected to be significantly higher in the ES+LF group compared to the LF or ES group (P<0.01). The concentration of urea was found to be higher in the LF group but only compared to the ES+LF group at day 28 (P<0.05) corresponding with a higher level of albumin in the LF group at the same collection day (P>0.05). The level of triglyceride decreased in the ES group (day 14-49). The concentration of glucose increased significantly in the LF group during the treatment (day 14, P<0.05) and also in the post-treatment phase compared to the control (day 28, 49, P<0.05). A lower level of aspartate aminotransferase was observed in both groups of dogs supplemented with E. senticosus extract compared to the control and LF group in the post-treatment period (ES, day 28, ES+LF group, day 49, P<0.05). Other parameters tested (albumin, cholesterol, alanine aminotransferase, glutathione peroxidase, calcium and phosphor) were not observed to change significantly (data not shown).

4. Discussion

E. senticosus does not belong to the group of plants with high antimicrobial potency, e.g. Origanum vulgare, Thymus vulgaris, Satureja hortensis etc. [19,20]. However, it contains chiisanogenin - a compound with broad but moderate inhibitory activities towards Gram-positive (staphylococci, Bacillus cereus) and also Gram-negative bacteria (E. coli, Samonella sp., Proteus sp., [21]). The poor antimicrobial effect of E. senticosus extract was reported also by Haviarova et al. [22] who assessed 330 bacterial indicators from which only 2.1% (staphylococci) were sensitive to the extract. The present in vitro assay shows that it is possible to combine E. senticosus extract with probiotic strain L. fermentum CCM 7421 since almost no growth inhibition was detected up to 2% (w/v) concentration in MRS broth. Dietary supplementation of dogs with L. fermentum CCM 7421 and E. senticosus extract (at a dose 8 mg/kg BW) did not negatively affect the colonisation and survival of the probiotic. The faecal persistance of the probiotic after cessation of application of the combined substances was similar, as in our previous experiments when the strain had been applied alone [7]. Microbiological analysis of faeces demonstrated no negative impact of E. senticosus extract administration on canine faecal microbiota in the

Exper. Time (d) P Value

Parameter

group 0 14 28 49 El5 Etb Ic

C 65.2 ± 2.1 65.0 ± 1.8 65.3 ± 1.3 64.9 ± 2.1

ES 65.0 ± 2.4 61.0 ± 2.2A 61.3 ± 2.6 61.9 ± 3.0

TP g/L 0.0044 0.9592 0.5902

LF 65.1 ± 1.2 63.8 ± 1.4A 64.9 ± 1.9 64.1 ± 1.6

ES + LF 64.9 ± 2.8 71.7 ± 2.0B 66.8 ± 2.4 68.3 ± 2.7

C 37.2 ± 0.9 37.1 ± 1.1 37.0 ± 1.0 37.1 ± 1.2

ES 37.3 ± 1.9 36.0 ± 1.1 36.9 ± 0.7 36.7 ± 1.4

ALB g/L 0.7350 0.9976 0.9992

LF 37.1 ± 0.9 37.9 ± 0.8 38.6 ± 0.9 38.1 ± 0.9

ES + LF 37.3 ± 2.8 37.4 ± 1.2 36.5 ± 3.3 37.4 ± 2.2

C 7.70 ± 0.61 7.74 ± 0.86 7.76 ± 0.50 7.72 ± 0.68

ES 7.58 ± 0.68 7.67 ± 0.55 7.68 ± 0.60 7.64 ± 0.58

UREA mmol/L 0.1016 0.9464 0.9324

LF 7.77 ± 0.57 7.86 ± 0.41 8.23 ± 0.52a 7.59 ± 0.41

ES + LF 7.60 ± 1.06 6.93 ± 0.76 5.98 ± 0.45b 6.90 ± 0.37

C 0.89 ± 0.12 0.90 ± 0.09 0.90 ± 0.06 0.92 ± 0.16

ES 0.89 ± 0.06 0.62 ± 0.05 0.60 ± 0.12 0.51 ± 0.08

TRIG mmol/L 0.0123 0.6675 0.8323

LF 0.91 ± 0.07 0.94 ± 0.13 0.98 ± 0.16 0.83 ± 0.13

ES + LF 0.90 ± 0.23 1.04 ± 0.14 0.84 ± 0.18 0.90 ± 0.18

C 4.58 ± 0.15 4.68 ± 0.12A 4.61 ± 0.22a 4.60 ± 0.15a

ES 4.55 ± 0.28 4.85 ± 0.49a 5.40 ± 0.37 5.27 ± 0.42 <0 0001

GLU mmol/L ^ и WU 1 0.0290 0.2610

LF 4.61 ± 0.31 5.99 ± 0.38Bb 5.75 ± 0.31Bb 5.66 ± 0.16b

ES + LF 4.40 ± 0.32 4.31 ± 0.09A 4.49 ± 0.23A 4.69 ± 0.12

C 0.21 ± 0.01 0.22 ± 0.02 0.21 ± 0.01a 0.21 ± 0.03a

ES 0.22 ± 0.06 0.15 ± 0.02 0.10 ± 0.02b 0.19 ± 0.03

AST ukat/L 0.0016 0.1783 0.1470

LF 0.22 ± 0.02 0.21 ± 0.02 0.22 ± 0.03a 0.23 ± 0.03A

ES + LF 0.21 ± 0.02 0.14 ± 0.03 0.17 ± 0.04 0.10 ± 0.02Bb

Table 4. Biochemical parameters in blood serum of control dogs (C, n=8), dogs fed E. senticosus (ES, n=8), L. fermentum CCM 7421 (LF, n=8) and the combination of both additives (ES+LF, n=8) for 14 days.

TP - total protein; ALB - albumin; TRIG - triglyceride; GLU - glucose; AST - aspartate aminotransferase.

Values are means ± SEM. Significant results: ab P<0.05; AB P<0.01; a Effect of treatment, b Effect of time; c Interaction.

dose range tested. Rather the opposite was observed: some regulative effects on abundance of gram-negative bacteria (detected mainly in the E. senticosus alone supplemented group) with no main effects on beneficial lactic acid bacteria (LAB) populations. Faecal numbers of LAB followed by probiotic application were detected to be significantly higher in both probiotic groups (LF, ES+LF) compared to the plant extract alone application (ES group). This agrees with a significant increase of lactobacilli observed in our canine study testing of 7-days application of L. fermentum CCM 7421 [7]. There is a lack of information on the pro/antimicrobial properties of E. senticosus studied under in vivo conditions, except for the experiment of Fang et al. [23] with weaned piglets which also indicated the potential of E. senticosus extract supplementation (0.1% of

diet) to regulate the intestinal microbiota composition. The reduction of staphylococcal population in faeces of rabbits after 3-weeks supplementation of diet with E. senticosus extract (0.015% of diet) was noted in our previous experiments [24]. The possible mechanism of E. senticosus regulative effects on microbiota could be of an immunological nature, for instance, by a modulation of lymphocytes, cytokines and antibodies which can mount an inhibitory defence against a wide range of bacteria [25]. The decrease in the numbers of clostridia detected in the LF group is a frequently observed result in canine studies after probiotic application [26,27]. However there are few studies that specifically investigate the mechanisms of Clostridium sp. colonisation resistance. Probiotics have been shown to block the attachment sites for clostridia or

their toxins and may directly destroy pathogenic toxins (e.g. toxin A or B of C. difficile; [28]). The stimulation of immune function (e.g. increase of IgA levels) is a further possible mechanism of anti-clostridial effect of probiotics [28]. A slight (significant only to ES group at day 14) but longer-lasting (day 7-49) increase of Aeromonas sp. and Pseudomonas sp. populations in the combined group might lead to an increased risk of translocation and infection, such as ear and skin infections, pneumonia or septicemia, more likely in immunocompromised animals.

Whereas the faecal microbiota was balanced, a no less important faecal characteristic - consistency - remained suboptimal throughout the study, except for the probiotic group (LF). The results revealed visually (FS) more liquid consistency in the ES+LF group (day 14, P<0.05), however a trend for lower DM content was observed in the ES group (day 49, P<0.05 compared to the LF group). The dose of E. senticosus extract or the length of its administration to dogs might be lower/ shorter from this viewpoint, but it remains to be tested. The concentration of faecal ammonia was partially higher only in the probiotic LF group at day 14 and 49 and may be connected with the lower faecal water content (higher DM), compared to the other treatments, as well as to the lower faecal pH that reduces ammonia absorption by its protonic dissociation (the formation of the less diffusible NH4+ compared to the diffusible NH3, [29]).

Furthermore, we studied the effects of used additives on the activation of blood macrophages which are known to participate in the immunological response by phagocytosis, antigen presentation and the production of cytokines, reactive oxygen species and nitrogen species involved in the destruction of pathogens [30]. Many investigators have demonstrated two contrary views on the immunomodulatory effects of E. senticosus: the stimulation [22,31,32] and the supression [33] of immune responses. It seems that the differing doses of adaptogen may play an important role in the immunomodulation effect, e.g. significant increase of phagocytosis were noted only at a dose rate of 0.030% extract in the diet compared to a nonsignificant increase at a dose rate of 0.015% in our rabbits experiments [22]. On the other hand, a lot of probiotic strains have an immunostimulation effect after their interaction with the M cells in Peyer's patches, with gut epithelial cells and with the associated immune cells. After contact with these cells, the release of cytokines is induced to up- or down-regulate the immune response [34]. The results of Donnet-Hughes et al. [35] suggest that a minimum daily dose of 109 viable L. johnsonii La1 was required to significantly modulate phagocytosis and respiratory burst activity and that faecal persistence

of the strain may not be a prerequisite for this form of immune reactivity. These authors detected significantly greater PA even 4 weeks after viable probiotic bacteria were no longer in the faeces. In our study, a significant increase of PA was detected 2 (P<0.001) and 5 (P<0.01) weeks after cessation of probiotic application, but the probiotic strain still persisted in certain levels in faeces. A significant increase of PA (P<0.05) after 4-days application of L. fermentum CCM 7421 strain was observed in our previous study using newborn Japanese quail [36]. The immunostimulation of the respiratory burst of the sole phagocytes is a strain-dependent characteristic of probiotics and mostly only long-term probiotic treatments have lead to significant results [37]. However, in our study a significant increase of respiratory burst activity (P<0.05) was noted only in the group treated with E. senticosus extract alone. To stimulate the immune status of dogs is of great importance in relation to the current alarming occurence of immunodeficiency caused by various factors such as aging, overvaccination, unproper nutrition (e.g. mycotoxins), certain drugs, stress etc. Interestingly, according to this study, unlike the probiotic the extract of E. senticosus seems to be an inappropriate additive to activate phagocytes in dogs.

In blood serum, the concentration of total protein was detected to be higher in dogs consuming only the combination of E. senticosus and L. fermentum CCM 7421 for 2 weeks (at day 14, P<0.01). Kong et al. [38] reported a significant increase of the serum contents and an apparent ileal digestibility of most amino acids tested in weaned piglets supplemented with E. senticosus extract (0.1% of diet). Although protein metabolism and nitrogen retention was shown to support also some probiotic strains in animals [39] including strain CCM 7421 but after a shorter supplementation period [7,36], no increase was detected in the LF group in this study. The possible reason of the specifity to increase total protein might be connected with differences in the microflora abundance (e.g. proteolytic genera) detected among treatments leading also to a different ammonia content in faeces and serum concentrations of urea. An effect of probiotic on serum protein level often depends on the length of its administration, on the initial serum protein concetrations (individual regulative effect of probiotic without changes in the average for the whole experiment) as well as on age of animals (growth support in the young age). Serum albumin concentration, a measure of nutritional status that tends to fall in many illnesses, was the highest, although non-significantly, in dogs supplemented with the probiotic alone (LF group, P>0.05). Whereas protein anabolism was stimulated after application of the probiotic and

E. senticosus combination, saccharide metabolism was supported especially in the probiotic group through the production of SCFA (the only group with a decrease of faecal pH, data on SCFA analysis not shown). An important effect of probiotics is activation of hepatic gluconeogenesis through lactic acid production [40]. An increase of glucose concentration in the LF group (day 14, P<0.05) can be explained by conversion of lactic acid to pyruvic acid and then to glucose. The glucose lowering effect of E. senticosus (syringin), observed in a few studies, appears to be related with the increase of insulin secretion [41] but was not indicated in our experiment. The decrease of triglyceride concentration was observed in the E. senticosus group of dogs during the treatment and also in the post-treatment phase. Several human studies deal with the influence of E. senticosus active components on serum lipid profile. Szolomicki et al. [41] observed a significant decrease of triglyceride, total cholesterol, LDL cholesterol and free fatty acids in healthy volunteers after 30 days treatment. Other authors detected a similar decrease of LDL cholesterol (P<0.001) whereas total cholesterol and triglyceride were not changed in blood serum of postmenopausal women supplemented with leaf extract for 6 months [10]. Although an inhibitory activity towards pancreatic lipase showed E. senticosus compounds [42], more detailed studies are needed to establish the exact mechanism of hypolipaemic action. Hepatoprotective effects are ascribed to certain E. senticosus components (e.g. glycoprotein) which are able to decrease elevated levels of alanine

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Acknowledgements

The study was financially supported by the Slovak Scientific Agency VEGA, project no. 2/0005/09. The help of Diploma student Jana Farbakova with dog handling is gratefully acknowledged.

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