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12 administration in healthy dogs
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Clinical microbiology
Effect of Bifidobacterium animalis B/12 administration in healthy dogs Q3
a Gancar Viola Strompfová a, *, Monika Pogány Simonová a, Son cíková b, b c c ová , Jana Farbáková , Aladár Mad’ari , Andrea Lauková a Dagmar Mudron Institute of Animal Physiology, Slovak Academy of Sciences, Soltésovej 4-6, 040 01 Kosice, Slovak Republic University of Veterinary Medicine and Pharmacy, Department of Microbiology and Immunology, Komenského 73, 041 81 Kosice, Slovakia c University of Veterinary Medicine and Pharmacy, Small Animals Clinic, Komenského 73, 041 81 Kosice, Slovakia a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 February 2014 Received in revised form 24 April 2014 Accepted 6 May 2014 Available online xxx
Bifidobacterium species constitute the most frequently used health-enhancing bacteria in functional foods or probiotic products, and most of their health benefits have been demonstrated in human or mice studies. However, knowledge of the effects of these bacteria in the canine organism is very limited. In this study, the canine-derived strain Bifidobacterium animalis B/12 (109 CFU) was tested for its effects on faecal microbiota, faecal characteristics, faecal organic acid concentrations, blood biochemistry, haematological and immunological parameters in healthy dogs (C-control, BA-B. animalis B/12 group, 10 dogs in each). The experiment lasted for 49 days with a 14-day treatment period (sample collection at days 0, 7, 14, 21, 28, and 49). A significantly higher population of lactic acid bacteria was detected (day 7) while the counts of coliform bacteria were lower in faeces of the BA group (days 14, 21, 28, 49) compared to control group C. Faecal concentrations of acetic (day 7, 21, 28, 49), acetoacetic (7e49) and valeric acid (14) were higher in contrast to formic acid (day 7e21), which was decreased after the treatment. In blood serum, significantly lower concentrations of triglyceride (day 14) and albumin (day 14, 28, 49) and significantly higher levels of alanine aminotransferase (day 14) and alkaline phosphatase (day 14, 28) were observed in the BA dogs. The phagocytic activity of leukocytes (especially of neutrophils) was higher in dogs after 14-day consumption of B/12 strain (day 14). The results show that many of these effects could also still be recorded several weeks after the treatment period. Ó 2014 Published by Elsevier Ltd.
Keywords: Bifidobacterium Probiotic Dogs Intestinal microbiota Blood biochemistry Haematology
1. Introduction The increasing interest in the intestinal microbiota includes mostly the study of genera/species composition in humans and animals, the impact of different factors (diet, stress, age, diseases) on this sensitive bacterial balance, as well as the study of beneficial bacteria supplementation on intestinal and overall health. Bifidobacteria (together with lactobacilli) are becoming the most studied bacteria because of many potential health benefits demonstrated especially in human trials. It seems, the genus Bifidobacterium sp. is not the predominant or stable component of canine gastrointestinal microbiota, as demonstrated in studies after the inclusion of molecular methods (16S rRNA gene pyrosequencing; [1,2]). Although faecal samples have commonly been used for analysis, Bifidobacterium sp. has not been recovered from all dogs, suggesting that the composition of microbiota is unique for each individual dog. Therefore also the species occurrence was variable from dog to * Corresponding author. Tel.: þ421 55 792 2974; fax: þ421 55 728 7842. E-mail address: [email protected] (V. Strompfová).
dog and from report to report, respectively; although Bifidobacterium animalis was more frequently identified bifidobacterial species in dogs [3e5]. Up to now, the strains of B. animalis, Bifidobacterium breve, Bifidobacterium longum and Bifidobacterium bifidum species are most frequently tested for the possible health benefits in infants or adults; however, the genus Bifidobacterium sp. comprises over 30 species (summarized [6]). The role of bifidobacteria in the intestinal microbiota is still limited, while the beneficial role for the host such as the prevention and treatment of viral diarrhoea, colon regularity (reduction in colonic transit), alleviation of lactose intolerance, cholesterol reduction, immunostimulatory effects and cancer prevention have been observed in many clinical studies [6,7]. The investigation of potential probiotic effects of bifidobacteria in dogs is only just beginning, and mostly includes only the results of microbiological analysis. For instance, the application of B. animalis AHC7 to dogs undergoing stress resulted in reduction of Clostridia and in optimal stool production compared with the control group and thus prevented stress-related gastrointestinal upsets and diarrhoea [8].
http://dx.doi.org/10.1016/j.anaerobe.2014.05.001 1075-9964/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Strompfová V, et al., Effect of Bifidobacterium animalis B/12 administration in healthy dogs, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.05.001
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Since there is lack of experiments studying the effects of bifidobacteria in dogs on the parameters such as organic acids in faeces, haematology, immunology or blood biochemistry, we investigated the impact of canine-derived strain B. animalis B/12 on these parameters. 2. Materials and methods 2.1. Animals and diet Healthy adult dogs (n ¼ 20; 12 males, 8 females) were randomly divided in two experimental groups, 10 animals in each (C e control, BA e B. animalis B/12 group). The age of the dogs ranged between 1 and 7 years (mean age 3.3 1.9) and body weights between 25.0 and 40.0 kg (mean BW 31.8 4.6 kg). They belonged to the following breeds: German Shepherd n ¼ 10, Belgian Shepherd (Malinois) n ¼ 7, cross-breed n ¼ 3. All experimental procedures were approved by the Ethics Commission of the Institute of Animal Physiology, Slovak Academy of Sciences (Kosice, Slovakia). The dogs were housed individually in a whole environmental but covered facility measuring 3.0 3.0 m with boxes 1.5 0.8 m (temperature, 4e10 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 (Happy Dog, Profi e Line Sportive, Wehringen, Germany), which contained (g/100 g diet): crude protein 26.0, crude fat 16.0, crude fibre 3.0, ash 6.5, calcium 1.6, phosphorus 0.95 (vitamin A 1025 IU, vitamin E 4.5 mg, vitamin D3 100 IU), 16.5 MJ metabolizable energy/kg. The adaptation period to this food was 4 weeks before the experiment. The experiment lasting 49 days was composed of baseline (day 0), supplementation (day 1e14) and postsupplementation periods (day 15e49). The animals were assigned to the control group (C, n ¼ 10) without any treatment and the BA group (n ¼ 10) supplemented with B. animalis B/12 (at a dose 1 mL of 1.04 109 CFU/ml of Ringer buffer, Merck). Dogs were administered the additive daily during the feeding time (for 14 days) and monitored for changes in body weight, clinical condition, vital parameters and appetite throughout the study. 2.2. Preparation of B. animalis B/12 for administration to dogs The strain B/12 was cultivated in MRS broth (Merck) supplemented with 0.3 g/L L-cysteine-HCl (SigmaeAldrich, USA)] under anaerobic conditions (Bactron Shel Lab II-1, Sheldon Manufacturing Inc., USA, atmosphere composition 90% N2 þ 5% H2 þ 5% CO2) at 37 C for 48 h. Cells were harvested after centrifugation (10 min at 2000 g) and culture sediment was resuspended in Ringer buffer (Merck, pH 7.0) to a concentration of 109 CFU/mL. The solution (1 mL) was pipetted into microtubes, covered with sunflower oil (200 mL) to avoid access of oxygen (the oil was removed before administration) and tubes were kept at 4 C. 2.3. 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 the faeces were correctly allocated to the proper animal. The determination of faecal score and pH measurement were performed 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 ml/mL of blood) for haematology, phagocytic activity and glutathione peroxidase determination, and (2) without anticoagulant for determination of biochemical parameters. The dogs were not allowed access to food in the 16-h overnight period prior to
venipuncture. The blood samples for testing of biochemical parameters were centrifuged (15 min at 2500 g) after 30 min and sera frozen at 20 C. 2.4. Microbiological analysis 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 using serial dilution (101e107). Aliquots of the dilutions (100 mL) were inoculated onto the following selective media: Mac Conkey agar to enumerate coliform bacteria (Becton and Dickinson, USA), M-Enterococcus agar for enterococci (Becton and Dickinson), MRS agar for lactic acid bacteria (LAB, Becton and Dickinson), TOS-propionate agar (Merck) for bifidobacteria, and Clostridium difficile agar base (Oxoid) for Clostridium-like bacteria. Plates were incubated aerobically at 37 C for 24e48 h. Clostridia and bifidobacteria were cultivated anaerobically at 38 C for 48e72 h. The results were expressed as log10 CFU per gram of moist faeces. 2.5. Faecal characteristics Fresh faecal samples were visually scored (FS-faecal score) according to the following system: 1 ¼ hard dry faeces; 2 ¼ hard, formed stool; 3 ¼ soft, formed, and moist stool; 4 ¼ soft, unformed stool; 5 ¼ watery liquid. Detection of pH (pH Metre, Hanna Instruments, USA) was performed immediately. Approximately 5 g of samples were stored at 20 until analysis of dry matter (DM). Ammonia concentrations was tested using Ammonia Assay Kit (SigmaeAldrich, USA). 2.6. Organic acids analysis Faecal samples (1 g) were diluted in 100 ml deionized water, homogenized (stomacher, IUL Instruments, Spain) and filtered through filter paper. A sample of 30 ml was used for analysis of SCFA (formic, lactic, succinic, acetic, acetoacetic, propionic, butyric, valeric acids) by capillary isotachophoresis (Isotachophoretic analyser ZKI 01, Radioecological Institute, Kosice, Slovakia). A leading electrolyte with the following composition was used in the preseparatory capillary: 102 M HCl þ 2.2 102 M ε-aminocaproic acid þ 0.1% methyl-hydroxyethylcellulosic acid, pH ¼ 4.3. A solution of 5 103 M caproic acid þ 2 102 M histidine was used as a finishing electrolyte. This electrolytic system worked at 250 mA in the pre-separatory and 50 mA in the analytic capillary. 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 (CA 590), inorganic phosphorus (PH 1016), glutathione peroxidase (RS 504) and alkaline phosphatase (ALP 120, BioLaTest, Erba Lachema, Czech Republic). Haematological parameters were analysed using the Cell-Dyn 3700 (Abbott Laboratories, USA). Phagocytic activity of monocytes and neutrophils was analysed using the Phagotest (ORPEGEN Pharma, Germany). This test is based on ingestion of FITC-labelled Escherichia coli bacteria by phagocytes. The results are expressed as the percentage of monocytes and granulocytes which ingested the labelled bacteria, and the mean fluorescence which correlates with the
Please cite this article in press as: Strompfová V, et al., Effect of Bifidobacterium animalis B/12 administration in healthy dogs, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.05.001
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3.2. Faecal characteristics
number of bacteria per individual leukocyte. Metabolic burst activity of phagocytes was measured with the FagoFlowEx kit (Exbio, Czech Republic) after stimulation of granulocytes with E. coli bacteria. The results are expressed as the stimulation index (SI), that is the ratio of the mean fluorescence intensity of positive granulocytes in the stimulated sample to that of negative granulocytes in the negative control. Stimulated granulocytes which proceed the oxidative burst exhibit bright fluorescence of rhodamine 123. Reactive intermediates which are produced during the oxidative burst inside phagocytes oxidize dihydrorhodamine 123 into fluorescent rhodamine 123 which was detected in a BDFACSCantoÔ flow cytometer (Becton Dickinson Biosciences, USA).
Almost no changes in pH values were detected during the treatment period (decrease by 0.2, P > 0.05) while the consistency of faeces was visibly more soft/liquid in the BA group compared to the control (faecal score higher by 0.3, day 7 and 14, P > 0.05, Table 1). The faecal dry mater content as well as the concentration of ammonia in faecal samples were not significantly affected (P > 0.05, Table 1). 3.3. Concentrations of organic acids in faeces
3. Results
The application of B. animalis B/12 led to a significant increase in acid concentrations (acetic, acetoacetic, valeric) in the treatment and also in the post-treatment period (acetic, acetoacetic acid, Table 2). The concentration of acetic acid was significantly higher at days 7, 21, 28 (P < 0.05) and 49 (P < 0.01) compared to the control dogs. An increase in acetoacetic acid was noted at days 7, 14 (P < 0.05), 21 (P < 0.01), 28 and 49 (P < 0.05). The concentration of valeric acid was significantly higher only at day 14 (P < 0.05). The total concentration of acids (lactic, acetic, propionic and butyric) was higher from day 7 to 49 (by 12e74 mmol/L, P > 0.05). In contrast, a decrease in formic acid concentration was noted at days 7, 14 (P < 0.01) and 21 (P < 0.05) in the BA group.
3.1. Microbial populations in faeces
3.4. Blood biochemistry and haematology
The addition of B. animalis B/12 to the diet of dogs resulted in a significantly higher population of lactic acid bacteria (LAB) in faeces during the application period (day 7, P < 0.05) compared to the control group (Table 1). The population of bifidobacteria was increased by only 0.6 log10 CFU/g during the supplementation period (day 7 and 14, P > 0.05). In contrast, the counts of Gram-negative bacteria were lower when compared to the control. In detail, the population of coliform bacteria was lower from day 14e49 in the BA group (P < 0.01 or P < 0.05 respectively, Table 1). Numbers of faecal Clostridiumlike bacteria and enterococci were not affected by the strain B/ 12 supplementation (P > 0.05).
In blood serum, the concentration of albumin was significantly lower at days 14, 28 (P < 0.01) and 49 (P < 0.05) in the BA group; however, the mean total protein concentration was not affected during the experiment (P > 0.05, Table 3). The concentration of triglyceride was lower in the BA group during the treatment (day 14, P < 0.05). The treatment effect was also observed in the activity of enzymes such as alanine aminotransferase (ALT) and alkaline phosphatase (ALP). A higher mean level of ALT was observed at day 14 (P < 0.05) while alkaline phosphatase was increased at day 14 and day 28 (P < 0.05). Other tested parameters (urea, cholesterol, glucose, aspartate aminotransferase, calcium and phosphor) were not changed significantly. Haematological parameters were not
2.8. Statistical analysis The results are expressed as the mean standard deviation. Statistical analyses were performed with GraphPad Prism software (version 5.0). Student’s unpaired t-test with the level of significance set at P < 0.05 was used to evaluate the control and the experimental group.
Table 1 Faecal microbial populations and faecal characteristics (mean standard deviation) of the control group (C, n ¼ 10) and the group fed Bifidobacterium animalis B/12 (BA, n ¼ 10) for 14 days. Microorganism/parameter
Group
Day 0
Lactic acid bacteria Bifidobacterium sp. Enterococcus sp. Clostridium-like sp. Coliform bacteria pH Faecal score Dry matter Ammonia (mg/g) Ammonia (mg/g DM) a,b
P < 0.05,
A,B
7
14 a
21
28
49
C BA C BA C BA C BA C BA
7.98 7.90 5.16 5.22 5.56 5.42 6.30 6.18 7.41 7.64
0.90 0.89 0.99 1.35 0.71 1.35 1.06 0.92 0.99 0.77
8.09 9.33 5.28 5.86 5.69 6.02 6.48 6.55 7.30 7.53
1.10 1.32b 1.14 1.40 0.97 1.18 0.68 1.08 1.03 1.44
7.90 7.04 5.36 5.90 5.50 4.67 6.58 6.64 7.52 5.87
0.85 1.64 1.02 0.76 1.26 1.11 1.01 1.26 0.90A 1.40B
8.10 7.54 5.30 5.12 5.27 4.69 6.32 6.47 7.60 5.84
1.21 1.08 0.79 0.65 0.70 0.86 0.83 1.43 0.89A 1.69B
8.23 7.80 5.29 5.44 5.55 5.31 6.41 7.43 7.42 6.16
0.76 0.90 0.66 0.75 1.13 0.74 0.77 1.04 1.21a 1.41b
8.15 7.36 5.16 5.47 5.49 5.66 6.21 6.27 7.02 5.71
0.98 0.92 0.78 1.15 0.89 0.81 0.64 1.33 0.65a 1.72b
C BA C BA C BA C BA C BA
5.90 5.91 2.80 2.75 28.1 28.6 0.71 0.74 2.80 2.71
0.18 0.28 0.30 0.56 1.9 3.9 0.17 0.26 0.83 1.10
5.85 5.66 2.78 3.10 28.6 29.2 0.73 0.81 2.89 2.85
0.33 0.49 0.41 0.30 2.3 6.7 0.21 0.26 0.68 0.99
5.92 5.67 2.81 3.00 28.1 27.8 0.71 0.81 2.91 3.01
0.41 0.36 0.22 0.32 4.0 2.9 0.30 0.42 1.04 1.71
5.81 5.79 2.81 2.75 27.9 27.7 0.75 0.65 2.74 2.39
0.51 0.73 0.38 0.34 1.1 2.7 0.32 0.26 0.90 0.99
5.88 5.79 2.78 2.89 28.2 28.9 0.75 0.78 2.86 2.73
0.31 0.22 0.41 0.52 2.4 3.2 0.22 0.36 1.08 1.22
5.86 6.16 2.70 2.88 28.0 27.5 0.70 0.73 2.79 2.68
0.36 0.28 0.26 0.54 1.9 2.6 0.39 0.18 0.69 0.70
P < 0.01.
Please cite this article in press as: Strompfová V, et al., Effect of Bifidobacterium animalis B/12 administration in healthy dogs, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.05.001
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Table 2 The concentration of organic acids (mean standard deviation) of the control dogs (C, n ¼ 6) and the group fed Bifidobacterium animalis B/12 (BA, n ¼ 10) for 14 days. Acid (mmol/L)
Group
Day 0
Lactic
C BA C BA C BA C BA C BA C BA C BA C BA C BA
Acetic Propionic Butyric Acetoacetic Succinic Valeric Formic SUMc
15.1 14.4 90.4 88.9 51.6 48.5 18.3 17.7 111.3 114.2 9.0 9.1 8.5 7.4 16.4 16.7 175.4 169.5
7
3.1 4.8 13.0 10.8 11.8 15.9 7.0 4.9 13.0 16.3 1.2 2.3 6.1 2.7 3.2 9.3 34.9 36.5
16.4 19.9 93.7 136.7 57.3 57.6 20.6 28.7 120.1 203.4 9.6 10.9 8.0 17.4 15.3 4.7 188.0 242.8
14
8.2 13.7 14.0a 44.3b 4.9 33.9 4.1 19.2 18.3a 70.6b 3.6 2.8 4.3a 6.6b 5.7A 0.7B 31.2 121.1
15.3 14.9 96.2 110.6 52.3 47.9 20.3 22.9 126.0 197.2 10.1 8.4 7.9 8.5 15.1 5.6 184.1 196.3
21
10.2 11.1 11.7 18.9 16.0 20.3 8.6 10.5 45.0a 40.9b 2.6 2.2 2.2 3.0 6.2A 0.5B 46.6 60.9
14.6 10.5 92.8 139.2 49.9 58.6 24.3 28.9 115.0 244.1 9.4 8.7 8.5 12.6 16.1 8.8 181.6 237.2
6.2 2.8 29.2a 27.0b 21.2 26.7 6.1 8.4 11.9A 70.5B 1.6 3.2 4.8 6.5 5.2a 3.2b 62.7 64.9
28
49
14.7 8.0 15.6 9.7 90.1 26.0a 135.9 25.2b 59.3 14.2 80.9 32.9 22.0 6.1 27.5 7.9 120.8 18.5a 201.3 78.2b 10.3 1.9 9.5 2.7 8.6 4.3 14.2 4.9 UD
15.3 9.2 16.4 7.8 92.2 16.9A 146.3 25.3B 60.0 26.8 64.6 18.9 20.9 4.6 23.7 5.4 120.0 48.3a 199.8 39.3b 10.1 3.2 10.9 2.8 9.2 3.6 15.8 6.5 UD
186.1 54.3 260.0 75.7
188.4 57.5 251.0 57.3
a,b
P < 0.05, A,BP < 0.01; UD e undetectable. SUM average of lactic, acetic, propionic and butyric acid concentration.
c
difference in the index of PA of monocytes, which was higher at day 14 (P < 0.05) in the BA group. The metabolic burst activity of granulocytes did not differ between the groups (P > 0.05, Table 5).
influenced significantly (Table 4) except for the concentration of haemoglobin, which was lower at days 14, 28 and 49 (P < 0.01) in the BA group. Eosinophil counts were decreased with significant difference at day 49 compared to control (P < 0.05).
4. Discussion 3.5. Phagocytic and metabolic burst activity of leukocytes 4.1. Characterization of B. animalis B/12 strain The total phagocytic activity (PA) of leukocytes was significantly higher (by over 10%) in the dogs after 14-day consumption of B. animalis B/12 (day 14, P < 0.01, Table 5); however, this was not so in the post-treatment period (day 28 and 49, P > 0.05). To see the results of cellular fraction of leukocytes, the PA of neutrophils was increased significantly (day 14, P < 0.01) compared to no visible differences in PA of monocytes (P > 0.05). There was significant
B. animalis B/12 was isolated from the faeces of a healthy dog (German Shepherd, female, 2 years old). In vitro, this strain was sensitive to most of the tested antimicrobial agents and showed inhibitory activity against Gram-negative bacteria (Enterobacter sp., E. coli, Klebsiella sp., Salmonella sp., Citrobacter sp.) based on production of organic acids [5]. B. animalis B/12 ferments 23/49 various
Table 3 Blood serum biochemical parameters (mean standard deviation) of the control group (C, n ¼ 10) and the group fed Bifidobacterium animalis B/12 (BA, n ¼ 10) for 14 days. Parameter
Group
Total protein (g/L)
C BA C BA C BA C BA C BA C BA C BA C BA C BA C BA C BA C BA
Day 0
Albumin (mmol/L) Urea (mmol/L) Triglyceride (mmol/L) Cholesterol (mmol/L) Glucose (mmol/L) Alanine aminotransferase (mkat/L) Aspartate aminotransferase (mkat/L) Alkaline Phosphatase (mkat/L) Glutathione peroxidase (mkat/L whole blood) Calcium (mmol/L) Phosphor (mmol/L) a,b
P < 0.05,
A,B
61.2 61.4 37.4 37.6 7.52 7.62 0.87 0.89 6.01 6.03 5.30 5.28 0.220 0.213 0.243 0.243 0.99 0.98 712 709 2.39 2.47 1.65 1.68
14
2.1 2.9 1.1 1.3 0.96 1.31 0.06 0.09a 0.98 1.28 0.27 0.31 0.088 0.048 0.046 0.068 0.36 0.27 69 76 0.18 0.16 0.07 0.12
61.2 61.0 37.6 34.9 7.47 7.23 0.88 0.82 5.98 5.77 5.28 5.12 0.203 0.329 0.251 0.383 1.01 1.38 718 758 2.41 2.40 1.63 1.61
28
2.8 3.6 1.5A 1.3B 1.14 1.00 0.06a 0.03b 1.14 1.00 0.17 0.19 0.030a 0.187b 0.095 0.178 0.21a 0.49b 81 106 0.13 0.20 0.10 0.09
61.0 61.1 37.3 35.6 7.37 7.38 0.87 0.84 5.72 5.68 5.30 5.20 0.210 0.214 0.246 0.270 1.01 1.45 710 701 2.39 2.40 1.64 1.64
49
1.9 2.8 1.1A 1.4B 1.02 1.13 0.06 0.04 1.03 1.30 0.24 0.36 0.068 0.063 0.061 0.058 0.25a 0.61b 65 81 0.26 0.16 0.09 0.12
61.3 62.0 37.5 36.3 7.49 7.80 0.87 0.83 6.03 6.05 5.28 5.28 0.206 0.259 0.241 0.246 1.06 1.30 714 758 2.38 2.37 1.63 1.65
2.1 2.0 1.1a 1.3b 0.93 0.77 0.07 0.04 0.78 1.24 0.16 0.27 0.096 0.130 0.088 0.042 0.58 0.46 87 123 0.11 0.16 0.14 0.09
P < 0.01.
Please cite this article in press as: Strompfová V, et al., Effect of Bifidobacterium animalis B/12 administration in healthy dogs, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.05.001
Q2
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Table 4 Haematological parameters (mean standard deviation) of the control group (C, n ¼ 10) and the group fed Bifidobacterium animalis B/12 (BA, n ¼ 10) for 14 days. Parameter
Group
Day 0
Red blood cells (G/L)
C BA C BA C BA C BA C BA C BA C BA C BA C BA
White blood cells (T/L) Haemoglobin (g/L) Haematocrit (L/L) Neutrophils (G/L) Lymphocytes (G/L) Monocytes (G/L) Eosinophils (G/L) Basophils (G/L) a,b
P < 0.05,
A,B
14
7.08 7.12 8.45 8.41 203.1 206.2 0.501 0.496 5.02 4.89 1.81 1.88 0.890 0.893 0.590 0.669 0.076 0.076
0.41 0.54 2.80 2.24 9.9 15.8 0.029 0.038 1.50 1.26 0.65 0.54 0.261 0.326 0.251 0.736 0.021 0.032
4.2. Faecal microflora and organic acid profile Consumption of B. animalis B/12 by dogs for 2 weeks led to a significant increase in LAB population (P < 0.05). The counts of bifidobacteria and enterococci were not influenced as well as the counts of not specified Clostridium-like bacteria. In contrast, the counts of coliforms were lower during and after treatment (P < 0.05). Almost no increase in faecal bifidobacteria is in accord with the canine study after consumption of B. animalis AHC7 for 6 weeks [9]. Despite the use of high doses of bifidobacteria (109 CFU) it seems that the adult canine intestinum at least is not a suitable environment for their colonization or multiplication (faecal population was up to 106 CFU/g). This opinion is also supported by the fact that bifidobacteria could not be isolated from all individual dogs [1,2]. Inhibitory effect of B. animalis B/12 against Gramnegative bacteria observed in vitro was also demonstrated in this
Table 5 Phagocytic activity and metabolic burst activity of leukocytes (mean standard deviation) measured in the control group (C, n ¼ 10) and in the group fed Bifidobacterium animalis B/12 (BA, n ¼ 10) for 14 days. Group
Day 0
FASUM FA Neu FA Mo IFASUM IFA Neu IFA Mo SI
0.59 0.64 2.10 2.37 10.0A 13.7B 0.031 0.037 2.11 1.82 0.50 0.68 0.301 0.382 0.462 0.425 0.044 0.063
7.10 7.29 8.57 9.04 203.9 187.7 0.506 0.508 5.60 6.06 1.78 1.68 0.810 0.810 0.507 0.451 0.067 0.072
49
0.39 0.68 1.90 2.92 12.5A 11.9B 0.041 0.043 2.08 3.05 0.62 0.35 0.296 0.294 0.370 0.543 0.032 0.047
7.12 7.16 8.60 9.62 199.3 182.1 0.502 0.498 5.20 6.60 1.73 1.92 0.834 0.856 0.430 0.162 0.073 0.086
0.55 0.71 2.90 2.70 8.9A 14.8B 0.039 0.044 1.99 2.29 0.49 0.50 0.670 0.361 0.300a 0.141b 0.026 0.038
P < 0.01.
carbohydrates with moderate b-galactosidase activity and survives in the presence of 0.3% bile and in artificial gastric juice (pH 2.5) sufficiently (decrease only by 0.5 log10 CFU/mL after 180 min, [5]).
Parameter
7.10 7.34 8.60 9.33 201.1 184.1 0.499 0.511 5.38 6.11 1.62 1.79 0.810 0.771 0.530 0.477 0.076 0.074
28
C BA C BA C BA C BA C BA C BA C BA
64.8 65.7 61.0 61.2 90.1 88.8 5043 5007 4280 4389 7111 7228 40.6 41.1
14
2.20 3.4 1.9 2.8 4.8 8.2 980 1359 655 972 810 1355 9.4 15.3
65.2 78.5 62.5 72.9 89.8 92.3 5001 5933 4870 5100 7200 8173 40.1 36.1
28
A
3.0 2.1B 2.3A 1.9B 2.8 3.6 1340 681 455 488 643a 915b 8.8 11.1
65.0 62.7 61.4 57.8 90.2 89.0 5021 5450 4970 4822 7577 7452 39.0 29.9
49
3.1 7.4 3.0 7.1 3.0 5.0 450 778 321 685 932 1203 12.0 10.8
63.7 65.7 62.9 64.9 90.2 90.4 4989 4764 4823 4643 7122 6640 39.0 41.3
2.4 6.2 2.8 7.4 1.9 2.4 541 882 630 821 805 807 5.1 6.9
FA e phagocytic activity; IFA e index of phagocytic activity; SI e stimulation index; Neu e neutrophils; Mo e monocytes. a,b P < 0.05, A,BP < 0.01.
in vivo study. The faecal coliforms were reduced from the end of the application period (day 14) till the end of the experiment (5 weeks after cessation of supplementation). Concurrently, significantly higher concentrations of acetic, valeric and acetoacetic acid were detected in the faeces. However, antimicrobial activity of lactobacilli and bifidobacteria against Gram-negative pathogenic bacteria is well known [10], although the mode of action of organic acid in the animal digestive tract is not fully understood. It has been assumed that undissociated forms of organic acids penetrate the lipid membrane of the bacterial cell and dissociate within the cell. As bacteria maintain the neutral pH of the cytoplasm, the export of excess protons consumes cellular ATP and results in depletion of energy [11]. The major end product of carbohydrate metabolism in bifidobacteria is acetate (B. animalis B/12 lactate:acetate 1:2.6 in vitro) which has higher pKa in comparison with lactic acid and therefore higher toxicity [12]. Since the proportion of undissociated molecules is dependent on pH values, the mean pH value during the treatment period was low enough (5.7, day 7 and 14) to exert a dissociative effect. A further mechanism of bacterial growth inhibition by acetic acids was proposed currently through genes expression control (involved in cellular metabolism and in the antiinflammatory response) or through the prevention of transepithelial electrical resistance reduction of the colonic epithelium, thereby improving intestinal defence [13]. The antimicrobial spectrum of acetate is very broad and in some cases more effective than common antiseptic solutions, e.g. against Pseudomonas aeruginosa [14]. Concerning the changes in acid concentrations, different results were obtained after application of Lactobacillus fermentum CCM 7421 to dogs, when butyric, succinic, formic and valeric acids were increased significantly [15]. 4.3. Faecal characteristics In our experiment, the faecal pH was decreased only by 0.2 (from 5.9 to 5.7, P > 0.05). It is similar to a value found in the canine experiment after supplementation with L. fermentum CCM 7421 [15]. Faecal consistency was visibly softer after the administration of B. animalis B/12 (increase by 0.3, P > 0.05); however, dry matter was almost not changed (difference up to 0.7%; P > 0.05). Sunvold et al. reported that faecal consistency score is more indicative of faecal characteristics than dry matter (poor correlations between faecal dry matter and faecal consistency exist [16]). Whereas the
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addition of beneficial LAB to dogs with diarrhoea resulted in more solid faecal consistency [17], it seems the addition of the same doses of probiotic bacteria to healthy dogs (with balanced intestinal microbiota) leads vice versa to more liquid faeces. 4.4. Blood biochemistry In blood serum, decrease in albumin (P < 0.05) and triglyceride (P < 0.05) concentrations and increase in ALT (P < 0.05) and ALP (P < 0.05) was observed compared with control dogs. The concentration of albumin fraction decreased significantly from day 14 till the end of the study (by up to 2.7 g/L); however, the total protein content was not changed. Albumin, a measure of nutritional status, is the most osmotically active plasma protein responsible for about 75% of colloid oncotic pressure. A major metabolic function of albumin is general binding and transport [18]. Inflammation and malnutrition reduce albumin concentrations by decreasing its rate of synthesis and accelerating its degradation [19]. To outline the link between albumin and diarrhoea occurrence, every 1 g/L increase in albumin concentration was associated with an 18% reduction in the odds of C. difficile associated diarrhoea [20]. Despite a decrease of albumin in our case, no dog had an albumin level below 33 g/L during the experiment, and thus no hypoalbuminaemia was observed. These results are contrary to our previous experiments using L. fermentum CCM 7421, where elevation by 1.5 g/L was detected [15]. Bifidobacteria or other LAB are often studied for lipid-lowering effects and reduction of body weight in human or mice experiments [21]. In this canine experiment, the mean body weight of the dogs was almost without change (decrease by 0.3 kg, day 14, P > 0.05). The concentration of triglyceride was lower at the end of treatment (P < 0.05), whereby cholesterol level was not reduced despite an increase of acetate concentrations, the primary substrate for cholesterol synthesis, [22]. On the other hand, some short-chain fatty acids (SCFA, e.g. propionate) decrease lipid concentrations by reducing acetate utilization for fatty acid and cholesterol synthesis in the liver. Since 90e95% of SCFA are rapidly absorbed in the large intestine the real proportion of SCFA absorbed into the blood does not reflect faecal concentrations [23]. Concerning serum transaminases, some elevation of both tested enzymes (ALT, AST) was detected during the treatment, followed by a decrease in the post-application period. One dog had the activity of AST and ALT over 0.7 at day 14, but this had decreased by the next sampling (day 28, 49). The available evidence suggests that some probiotic strains including Bifidobacterium sp. are capable of preventing gut permeability, bacterial translocation, endotoxemia (associated with reduction of hepatic enzyme activity) and pro-inflammatory cytokine production in liver injury models [24]. In our case, liver activity was increased, most probably by metabolically active SCFA; it seems, this effect also depends on the health status of the animal. Most studies including experiments with the application of L. fermentum CCM 7421 to dogs have demonstrated reduction in hepatic enzyme activity in individuals with liver damage (e.g. inflammatory bowel disease, alcohol-induced liver injury, [17,24]. The activity of ALP was also significantly higher from day 14 compared to control, and could indicate intestinal or tissue (liver, bones, kidneys) activity or damage. Reinforced activity of bone tissue cells could balance the acidifying effect of resorbed SCFA (to retain optimal serum pH and mineral levels). 4.5. Haematology and phagocytic activity of leukocytes In this study, significant decrease in haemoglobin concentration (P < 0.01, within the limits of the physiological range) was noted at day 14 and 2e5 weeks after cessation of supplementation; however,
the red blood cell count was not changed. The decrease in haemoglobin could again be connected with the fermentation metabolites (mostly SCFA) and the diet composition (a larger amount of carbohydrates produces a larger amount of SCFA). Recently, increasing evidence for haemoglobin reducing effect caused by LAB exists [25,26]; therefore, it is necessary to test haematological parameters which have often been omitted in animal studies. Concerning the effect of B. animalis B/12 on the activation of blood macrophages, the total PA of leukocytes (P < 0.01) and PA of neutrophils (P < 0.01) were significantly higher during the supplementation phase (day 14) compared to control. The index of PA was increased significantly only in monocytes (P < 0.05). Significant stimulation of PA in granulocytes is consistent with our previous canine study [15]; however, the time of PA increase and the length of this increase differed (the same experimental design was used). Whereas PA increased progressively after application of L. fermentum CCM 7421 with the significant difference in the post-treatment period (day 49), B. animalis B/12 resulted in immediate increase of PA during the treatment (day 14) with decrease to basal values in the post-treatment period. While phagocytosis increased, respiratory burst activity tended to decrease (day 14, 28) with no significant difference. Although the exact mode of stimulation is not well understood, phagocytic cells are probably stimulated after direct contact with LAB or sub-cellular fractions of bacterial cell walls. They can induce mononuclear leukocytes to secrete procellular immunity cytokines, which serves to drive the immune system towards a general enhancement of cellular immune function [27]. In conclusion, this study demonstrates that application of B. animalis B/12 to healthy dogs is associated with the reduction of coliform bacteria in faeces, decrease in haemoglobin, albumin and triglyceride serum concentrations and increase in ALT, ALP and PA of neutrophils. It seems that daily long-term applications of LAB in higher doses (over 108 CFU) to healthy dogs are not desirable. According to presented and our previous results, many of the effects lasted for several weeks after the treatment period. This means that some of the LAB effects are persistent in the long term. Acknowledgements The study was financially supported by the Slovak Scientific Agency VEGA, project no. 2/0056/13. The help of dogs owners is gratefully acknowledged. References [1] Greetham HL, Giffard C, Hutson RA, Collins MD, Gibson GR. Bacteriology of the Labrador dog gut: a cultural and genotypic approach. J Appl Microbiol 2002;93:640e6. [2] Handl S, Dowd SE, Garzia-Mazcorro JF, Steiner JM, Suchodolski JS. Massive parallel 16S rRNA gene pyrosequencing reveals highly diverse fecal bacterial and fungal communities in healthy dogs and cats. FEMS Microbiol Ecol 2011;76:301e10. [3] Kim SY, Adachi Y. Biological and genetic classification of canine intestinal lactic acid bacteria and bifidobacteria. Microbiol Immunol 2007;51:919e28. Svobodová I, Jebavý L, et al. Bifi[4] Bunesová V, Vlková E, Rada V, Ro cková S, dobacterium animalis subsp. lactis strains isolated from dog faeces. Vet Microbiol 2012;160:501e5. [5] Strompfová V, Lauková A. Isolation and characterization of faecal bifidobacteria and lactobacilli isolated from dogs and primates. Anaerobe; 2014. http:// dx.doi.org/10.1016/j.anaerobe.2013.10.007. [6] Lee J-H, O’Sullivan DJ. Genomic insights into bifidobacteria. Microbiol Mol Biol Rev 2010;74:378e416. [7] Picard C, Fioramonti J, Francois A, Robinson T, Neant F, Matuchansky C. Review article: bifidobacteria as probiotic agents e physiological effects and clinical benefits. Aliment Pharmacol Ther 2005;22:495e512. [8] Kelley R, Levy K, Mundell P, Hayek MG. Effects of varying doses of a probiotic supplement fed to healthy dogs undergoing kenneling stress. Int J Appl Res Vet Med 2012;10:205e16. [9] ÓMahony D, Barry Murphy K, MacSharry J, Boileau T, Sunvold G, Reinhart G, et al. Portrait of a canine probiotic Bifidobacterium e from gut to gut. Vet Microbiol 2009;139:106e12.
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V. Strompfová et al. / Anaerobe xxx (2014) 1e7 } tt P, Shchepetova J, Lõivukene K, Kullisaar T, Mikelsaar M. Antagonistic [10] Hu activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens. J Appl Microbiol 2006;100:1324e32. [11] Ricke SC. Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Poult Sci 2003;82:632e9. [12] Vázquez JA, Durán A, Rodríguez-Amado I, Prieto MA, Rial D, Murado MA. Evaluation of toxic effects of several carboxylic acids on bacterial growth by toxicodynamic modelling. Microb Cell Fact 2011;10:100. [13] Fukuda S, Toh H, Hase K, Oshima K, Nakanishi Y, Yoshimura K, et al. Bifidobacteria can protect from enteropathogenic infection through production of acetate. Nature 2011;469:543e7. [14] Ryssel H, Kloeters O, Germann G, Schäfer Th, Wiedemann G, Oehlbauer M. The antimicrobial effect of acetic acid e an alternative to common local antiseptics? Burns 2009;35:695e700. ová D, Lauková A. [15] Strompfová V, Plachá I, Cobanová K, Gancar cíková S, Mudron Experimental addition of Eleutherococcus senticosus and probiotic to the canine diet. Cent Eur J Biol 2012;7:436e47. [16] Sunvold GD, Fahey Jr GC, Merchen NR, Titgemeyer EC, Bourquin LD, Bauer LL, et al. Dietary fiber for dogs: IV. In vitro fermentation of selected fiber sources by dog fecal inoculum and in vivo digestion and metabolism of fibersupplemented diets. J Anim Sci 1995;73:1099e109. áková M, Simonová M, Lauková A, Fialkovi [17] Strompfová V, Marcin cová M. Probiotic strain Lactobacillus fermentum CCM 7421, canine isolate applied to dogs suffering from gastrointestinal disorders. Int J Prob Preb 2007;2:233e8. [18] McGrotty Y, Knottenbelt C. Significance of plasma protein abnormalities in dogs and cats. Practice 2002;24:512e7.
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[19] Don BR, Kaysen G. Serum albumin: relationship to inflammation and nutrition. Semin Dial 2004;17:432e7. [20] Hickson M, D’Souza AL, Muthu N, Rogers TR, Want S, Rajmkumar C, et al. Use of probiotic Lactobacillus preparation to prevent diarrhoea associated with antibiotics: randomised double blind placebo controlled trial. BMJ 2007;335: 80. [21] Cano PG, Santacruz A, Trejo FM, Sanz Y. Bifidobacterium CECT 7765 improves metabolic and immunological alterations associated with obesity in high-fat diet-fed mice. Obesity 2013;21:2310e21. [22] Pereira DIA, Gibson GR. Effects of consumption of probiotics and prebiotics on serum lipid levels in humans. Crit Rev Biochem Mol Biol 2002;37:259e81. [23] Hijova E, Chmelarova A. Short chain fatty acids and colonic health. Bratisl-Lek Listy 2007;108:354e8. [24] Kirpich IA, Solovieva NV, Leikhter SN, Shidakova NA, Lebedeva OV, Sidorov PI, et al. Probiotics restore bowel flora and improve liver enzymes in human alcohol-induced liver injury: a pilot study. Alcohol 2008;42:675e82. [25] Uchida M, Tsuboi H, Takahashi M, Nemoto A, Seki K, Tsunoo H, et al. Safety of high doses of Propionibacterium freundenreichii ET-3 culture in healthy adult subjects. Reg Toxicol Pharmacol 2011;60:262e7. [26] Dahyia T, Sihag RC, Gahlawat SK. Effect of probiotics on the haematological parameters of Indian Magur (Clarius batrachus L.). J Fisch Aquat Sci; 2012: 279e90. [27] Chiang BL, Sheih YH, Wang LH, Liao CK, Gill HS. Enhancing immunity by dietary consumption of a probiotic lactic acid bacterium (Bifidobacterium lactis HN019): optimization and definition of cellular immune responses. Eur J Clin Nutr 2000;54:849e55.
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Clinical microbiology
Effect of Bifidobacterium animalis B/12 administration in healthy dogs Q3
a Gancar Viola Strompfová a, *, Monika Pogány Simonová a, Son cíková b, b c c ová , Jana Farbáková , Aladár Mad’ari , Andrea Lauková a Dagmar Mudron Institute of Animal Physiology, Slovak Academy of Sciences, Soltésovej 4-6, 040 01 Kosice, Slovak Republic University of Veterinary Medicine and Pharmacy, Department of Microbiology and Immunology, Komenského 73, 041 81 Kosice, Slovakia c University of Veterinary Medicine and Pharmacy, Small Animals Clinic, Komenského 73, 041 81 Kosice, Slovakia a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 February 2014 Received in revised form 24 April 2014 Accepted 6 May 2014 Available online xxx
Bifidobacterium species constitute the most frequently used health-enhancing bacteria in functional foods or probiotic products, and most of their health benefits have been demonstrated in human or mice studies. However, knowledge of the effects of these bacteria in the canine organism is very limited. In this study, the canine-derived strain Bifidobacterium animalis B/12 (109 CFU) was tested for its effects on faecal microbiota, faecal characteristics, faecal organic acid concentrations, blood biochemistry, haematological and immunological parameters in healthy dogs (C-control, BA-B. animalis B/12 group, 10 dogs in each). The experiment lasted for 49 days with a 14-day treatment period (sample collection at days 0, 7, 14, 21, 28, and 49). A significantly higher population of lactic acid bacteria was detected (day 7) while the counts of coliform bacteria were lower in faeces of the BA group (days 14, 21, 28, 49) compared to control group C. Faecal concentrations of acetic (day 7, 21, 28, 49), acetoacetic (7e49) and valeric acid (14) were higher in contrast to formic acid (day 7e21), which was decreased after the treatment. In blood serum, significantly lower concentrations of triglyceride (day 14) and albumin (day 14, 28, 49) and significantly higher levels of alanine aminotransferase (day 14) and alkaline phosphatase (day 14, 28) were observed in the BA dogs. The phagocytic activity of leukocytes (especially of neutrophils) was higher in dogs after 14-day consumption of B/12 strain (day 14). The results show that many of these effects could also still be recorded several weeks after the treatment period. Ó 2014 Published by Elsevier Ltd.
Keywords: Bifidobacterium Probiotic Dogs Intestinal microbiota Blood biochemistry Haematology
1. Introduction The increasing interest in the intestinal microbiota includes mostly the study of genera/species composition in humans and animals, the impact of different factors (diet, stress, age, diseases) on this sensitive bacterial balance, as well as the study of beneficial bacteria supplementation on intestinal and overall health. Bifidobacteria (together with lactobacilli) are becoming the most studied bacteria because of many potential health benefits demonstrated especially in human trials. It seems, the genus Bifidobacterium sp. is not the predominant or stable component of canine gastrointestinal microbiota, as demonstrated in studies after the inclusion of molecular methods (16S rRNA gene pyrosequencing; [1,2]). Although faecal samples have commonly been used for analysis, Bifidobacterium sp. has not been recovered from all dogs, suggesting that the composition of microbiota is unique for each individual dog. Therefore also the species occurrence was variable from dog to * Corresponding author. Tel.: þ421 55 792 2974; fax: þ421 55 728 7842. E-mail address: [email protected] (V. Strompfová).
dog and from report to report, respectively; although Bifidobacterium animalis was more frequently identified bifidobacterial species in dogs [3e5]. Up to now, the strains of B. animalis, Bifidobacterium breve, Bifidobacterium longum and Bifidobacterium bifidum species are most frequently tested for the possible health benefits in infants or adults; however, the genus Bifidobacterium sp. comprises over 30 species (summarized [6]). The role of bifidobacteria in the intestinal microbiota is still limited, while the beneficial role for the host such as the prevention and treatment of viral diarrhoea, colon regularity (reduction in colonic transit), alleviation of lactose intolerance, cholesterol reduction, immunostimulatory effects and cancer prevention have been observed in many clinical studies [6,7]. The investigation of potential probiotic effects of bifidobacteria in dogs is only just beginning, and mostly includes only the results of microbiological analysis. For instance, the application of B. animalis AHC7 to dogs undergoing stress resulted in reduction of Clostridia and in optimal stool production compared with the control group and thus prevented stress-related gastrointestinal upsets and diarrhoea [8].
http://dx.doi.org/10.1016/j.anaerobe.2014.05.001 1075-9964/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Strompfová V, et al., Effect of Bifidobacterium animalis B/12 administration in healthy dogs, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.05.001
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Since there is lack of experiments studying the effects of bifidobacteria in dogs on the parameters such as organic acids in faeces, haematology, immunology or blood biochemistry, we investigated the impact of canine-derived strain B. animalis B/12 on these parameters. 2. Materials and methods 2.1. Animals and diet Healthy adult dogs (n ¼ 20; 12 males, 8 females) were randomly divided in two experimental groups, 10 animals in each (C e control, BA e B. animalis B/12 group). The age of the dogs ranged between 1 and 7 years (mean age 3.3 1.9) and body weights between 25.0 and 40.0 kg (mean BW 31.8 4.6 kg). They belonged to the following breeds: German Shepherd n ¼ 10, Belgian Shepherd (Malinois) n ¼ 7, cross-breed n ¼ 3. All experimental procedures were approved by the Ethics Commission of the Institute of Animal Physiology, Slovak Academy of Sciences (Kosice, Slovakia). The dogs were housed individually in a whole environmental but covered facility measuring 3.0 3.0 m with boxes 1.5 0.8 m (temperature, 4e10 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 (Happy Dog, Profi e Line Sportive, Wehringen, Germany), which contained (g/100 g diet): crude protein 26.0, crude fat 16.0, crude fibre 3.0, ash 6.5, calcium 1.6, phosphorus 0.95 (vitamin A 1025 IU, vitamin E 4.5 mg, vitamin D3 100 IU), 16.5 MJ metabolizable energy/kg. The adaptation period to this food was 4 weeks before the experiment. The experiment lasting 49 days was composed of baseline (day 0), supplementation (day 1e14) and postsupplementation periods (day 15e49). The animals were assigned to the control group (C, n ¼ 10) without any treatment and the BA group (n ¼ 10) supplemented with B. animalis B/12 (at a dose 1 mL of 1.04 109 CFU/ml of Ringer buffer, Merck). Dogs were administered the additive daily during the feeding time (for 14 days) and monitored for changes in body weight, clinical condition, vital parameters and appetite throughout the study. 2.2. Preparation of B. animalis B/12 for administration to dogs The strain B/12 was cultivated in MRS broth (Merck) supplemented with 0.3 g/L L-cysteine-HCl (SigmaeAldrich, USA)] under anaerobic conditions (Bactron Shel Lab II-1, Sheldon Manufacturing Inc., USA, atmosphere composition 90% N2 þ 5% H2 þ 5% CO2) at 37 C for 48 h. Cells were harvested after centrifugation (10 min at 2000 g) and culture sediment was resuspended in Ringer buffer (Merck, pH 7.0) to a concentration of 109 CFU/mL. The solution (1 mL) was pipetted into microtubes, covered with sunflower oil (200 mL) to avoid access of oxygen (the oil was removed before administration) and tubes were kept at 4 C. 2.3. 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 the faeces were correctly allocated to the proper animal. The determination of faecal score and pH measurement were performed 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 ml/mL of blood) for haematology, phagocytic activity and glutathione peroxidase determination, and (2) without anticoagulant for determination of biochemical parameters. The dogs were not allowed access to food in the 16-h overnight period prior to
venipuncture. The blood samples for testing of biochemical parameters were centrifuged (15 min at 2500 g) after 30 min and sera frozen at 20 C. 2.4. Microbiological analysis 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 using serial dilution (101e107). Aliquots of the dilutions (100 mL) were inoculated onto the following selective media: Mac Conkey agar to enumerate coliform bacteria (Becton and Dickinson, USA), M-Enterococcus agar for enterococci (Becton and Dickinson), MRS agar for lactic acid bacteria (LAB, Becton and Dickinson), TOS-propionate agar (Merck) for bifidobacteria, and Clostridium difficile agar base (Oxoid) for Clostridium-like bacteria. Plates were incubated aerobically at 37 C for 24e48 h. Clostridia and bifidobacteria were cultivated anaerobically at 38 C for 48e72 h. The results were expressed as log10 CFU per gram of moist faeces. 2.5. Faecal characteristics Fresh faecal samples were visually scored (FS-faecal score) according to the following system: 1 ¼ hard dry faeces; 2 ¼ hard, formed stool; 3 ¼ soft, formed, and moist stool; 4 ¼ soft, unformed stool; 5 ¼ watery liquid. Detection of pH (pH Metre, Hanna Instruments, USA) was performed immediately. Approximately 5 g of samples were stored at 20 until analysis of dry matter (DM). Ammonia concentrations was tested using Ammonia Assay Kit (SigmaeAldrich, USA). 2.6. Organic acids analysis Faecal samples (1 g) were diluted in 100 ml deionized water, homogenized (stomacher, IUL Instruments, Spain) and filtered through filter paper. A sample of 30 ml was used for analysis of SCFA (formic, lactic, succinic, acetic, acetoacetic, propionic, butyric, valeric acids) by capillary isotachophoresis (Isotachophoretic analyser ZKI 01, Radioecological Institute, Kosice, Slovakia). A leading electrolyte with the following composition was used in the preseparatory capillary: 102 M HCl þ 2.2 102 M ε-aminocaproic acid þ 0.1% methyl-hydroxyethylcellulosic acid, pH ¼ 4.3. A solution of 5 103 M caproic acid þ 2 102 M histidine was used as a finishing electrolyte. This electrolytic system worked at 250 mA in the pre-separatory and 50 mA in the analytic capillary. 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 (CA 590), inorganic phosphorus (PH 1016), glutathione peroxidase (RS 504) and alkaline phosphatase (ALP 120, BioLaTest, Erba Lachema, Czech Republic). Haematological parameters were analysed using the Cell-Dyn 3700 (Abbott Laboratories, USA). Phagocytic activity of monocytes and neutrophils was analysed using the Phagotest (ORPEGEN Pharma, Germany). This test is based on ingestion of FITC-labelled Escherichia coli bacteria by phagocytes. The results are expressed as the percentage of monocytes and granulocytes which ingested the labelled bacteria, and the mean fluorescence which correlates with the
Please cite this article in press as: Strompfová V, et al., Effect of Bifidobacterium animalis B/12 administration in healthy dogs, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.05.001
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3.2. Faecal characteristics
number of bacteria per individual leukocyte. Metabolic burst activity of phagocytes was measured with the FagoFlowEx kit (Exbio, Czech Republic) after stimulation of granulocytes with E. coli bacteria. The results are expressed as the stimulation index (SI), that is the ratio of the mean fluorescence intensity of positive granulocytes in the stimulated sample to that of negative granulocytes in the negative control. Stimulated granulocytes which proceed the oxidative burst exhibit bright fluorescence of rhodamine 123. Reactive intermediates which are produced during the oxidative burst inside phagocytes oxidize dihydrorhodamine 123 into fluorescent rhodamine 123 which was detected in a BDFACSCantoÔ flow cytometer (Becton Dickinson Biosciences, USA).
Almost no changes in pH values were detected during the treatment period (decrease by 0.2, P > 0.05) while the consistency of faeces was visibly more soft/liquid in the BA group compared to the control (faecal score higher by 0.3, day 7 and 14, P > 0.05, Table 1). The faecal dry mater content as well as the concentration of ammonia in faecal samples were not significantly affected (P > 0.05, Table 1). 3.3. Concentrations of organic acids in faeces
3. Results
The application of B. animalis B/12 led to a significant increase in acid concentrations (acetic, acetoacetic, valeric) in the treatment and also in the post-treatment period (acetic, acetoacetic acid, Table 2). The concentration of acetic acid was significantly higher at days 7, 21, 28 (P < 0.05) and 49 (P < 0.01) compared to the control dogs. An increase in acetoacetic acid was noted at days 7, 14 (P < 0.05), 21 (P < 0.01), 28 and 49 (P < 0.05). The concentration of valeric acid was significantly higher only at day 14 (P < 0.05). The total concentration of acids (lactic, acetic, propionic and butyric) was higher from day 7 to 49 (by 12e74 mmol/L, P > 0.05). In contrast, a decrease in formic acid concentration was noted at days 7, 14 (P < 0.01) and 21 (P < 0.05) in the BA group.
3.1. Microbial populations in faeces
3.4. Blood biochemistry and haematology
The addition of B. animalis B/12 to the diet of dogs resulted in a significantly higher population of lactic acid bacteria (LAB) in faeces during the application period (day 7, P < 0.05) compared to the control group (Table 1). The population of bifidobacteria was increased by only 0.6 log10 CFU/g during the supplementation period (day 7 and 14, P > 0.05). In contrast, the counts of Gram-negative bacteria were lower when compared to the control. In detail, the population of coliform bacteria was lower from day 14e49 in the BA group (P < 0.01 or P < 0.05 respectively, Table 1). Numbers of faecal Clostridiumlike bacteria and enterococci were not affected by the strain B/ 12 supplementation (P > 0.05).
In blood serum, the concentration of albumin was significantly lower at days 14, 28 (P < 0.01) and 49 (P < 0.05) in the BA group; however, the mean total protein concentration was not affected during the experiment (P > 0.05, Table 3). The concentration of triglyceride was lower in the BA group during the treatment (day 14, P < 0.05). The treatment effect was also observed in the activity of enzymes such as alanine aminotransferase (ALT) and alkaline phosphatase (ALP). A higher mean level of ALT was observed at day 14 (P < 0.05) while alkaline phosphatase was increased at day 14 and day 28 (P < 0.05). Other tested parameters (urea, cholesterol, glucose, aspartate aminotransferase, calcium and phosphor) were not changed significantly. Haematological parameters were not
2.8. Statistical analysis The results are expressed as the mean standard deviation. Statistical analyses were performed with GraphPad Prism software (version 5.0). Student’s unpaired t-test with the level of significance set at P < 0.05 was used to evaluate the control and the experimental group.
Table 1 Faecal microbial populations and faecal characteristics (mean standard deviation) of the control group (C, n ¼ 10) and the group fed Bifidobacterium animalis B/12 (BA, n ¼ 10) for 14 days. Microorganism/parameter
Group
Day 0
Lactic acid bacteria Bifidobacterium sp. Enterococcus sp. Clostridium-like sp. Coliform bacteria pH Faecal score Dry matter Ammonia (mg/g) Ammonia (mg/g DM) a,b
P < 0.05,
A,B
7
14 a
21
28
49
C BA C BA C BA C BA C BA
7.98 7.90 5.16 5.22 5.56 5.42 6.30 6.18 7.41 7.64
0.90 0.89 0.99 1.35 0.71 1.35 1.06 0.92 0.99 0.77
8.09 9.33 5.28 5.86 5.69 6.02 6.48 6.55 7.30 7.53
1.10 1.32b 1.14 1.40 0.97 1.18 0.68 1.08 1.03 1.44
7.90 7.04 5.36 5.90 5.50 4.67 6.58 6.64 7.52 5.87
0.85 1.64 1.02 0.76 1.26 1.11 1.01 1.26 0.90A 1.40B
8.10 7.54 5.30 5.12 5.27 4.69 6.32 6.47 7.60 5.84
1.21 1.08 0.79 0.65 0.70 0.86 0.83 1.43 0.89A 1.69B
8.23 7.80 5.29 5.44 5.55 5.31 6.41 7.43 7.42 6.16
0.76 0.90 0.66 0.75 1.13 0.74 0.77 1.04 1.21a 1.41b
8.15 7.36 5.16 5.47 5.49 5.66 6.21 6.27 7.02 5.71
0.98 0.92 0.78 1.15 0.89 0.81 0.64 1.33 0.65a 1.72b
C BA C BA C BA C BA C BA
5.90 5.91 2.80 2.75 28.1 28.6 0.71 0.74 2.80 2.71
0.18 0.28 0.30 0.56 1.9 3.9 0.17 0.26 0.83 1.10
5.85 5.66 2.78 3.10 28.6 29.2 0.73 0.81 2.89 2.85
0.33 0.49 0.41 0.30 2.3 6.7 0.21 0.26 0.68 0.99
5.92 5.67 2.81 3.00 28.1 27.8 0.71 0.81 2.91 3.01
0.41 0.36 0.22 0.32 4.0 2.9 0.30 0.42 1.04 1.71
5.81 5.79 2.81 2.75 27.9 27.7 0.75 0.65 2.74 2.39
0.51 0.73 0.38 0.34 1.1 2.7 0.32 0.26 0.90 0.99
5.88 5.79 2.78 2.89 28.2 28.9 0.75 0.78 2.86 2.73
0.31 0.22 0.41 0.52 2.4 3.2 0.22 0.36 1.08 1.22
5.86 6.16 2.70 2.88 28.0 27.5 0.70 0.73 2.79 2.68
0.36 0.28 0.26 0.54 1.9 2.6 0.39 0.18 0.69 0.70
P < 0.01.
Please cite this article in press as: Strompfová V, et al., Effect of Bifidobacterium animalis B/12 administration in healthy dogs, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.05.001
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Table 2 The concentration of organic acids (mean standard deviation) of the control dogs (C, n ¼ 6) and the group fed Bifidobacterium animalis B/12 (BA, n ¼ 10) for 14 days. Acid (mmol/L)
Group
Day 0
Lactic
C BA C BA C BA C BA C BA C BA C BA C BA C BA
Acetic Propionic Butyric Acetoacetic Succinic Valeric Formic SUMc
15.1 14.4 90.4 88.9 51.6 48.5 18.3 17.7 111.3 114.2 9.0 9.1 8.5 7.4 16.4 16.7 175.4 169.5
7
3.1 4.8 13.0 10.8 11.8 15.9 7.0 4.9 13.0 16.3 1.2 2.3 6.1 2.7 3.2 9.3 34.9 36.5
16.4 19.9 93.7 136.7 57.3 57.6 20.6 28.7 120.1 203.4 9.6 10.9 8.0 17.4 15.3 4.7 188.0 242.8
14
8.2 13.7 14.0a 44.3b 4.9 33.9 4.1 19.2 18.3a 70.6b 3.6 2.8 4.3a 6.6b 5.7A 0.7B 31.2 121.1
15.3 14.9 96.2 110.6 52.3 47.9 20.3 22.9 126.0 197.2 10.1 8.4 7.9 8.5 15.1 5.6 184.1 196.3
21
10.2 11.1 11.7 18.9 16.0 20.3 8.6 10.5 45.0a 40.9b 2.6 2.2 2.2 3.0 6.2A 0.5B 46.6 60.9
14.6 10.5 92.8 139.2 49.9 58.6 24.3 28.9 115.0 244.1 9.4 8.7 8.5 12.6 16.1 8.8 181.6 237.2
6.2 2.8 29.2a 27.0b 21.2 26.7 6.1 8.4 11.9A 70.5B 1.6 3.2 4.8 6.5 5.2a 3.2b 62.7 64.9
28
49
14.7 8.0 15.6 9.7 90.1 26.0a 135.9 25.2b 59.3 14.2 80.9 32.9 22.0 6.1 27.5 7.9 120.8 18.5a 201.3 78.2b 10.3 1.9 9.5 2.7 8.6 4.3 14.2 4.9 UD
15.3 9.2 16.4 7.8 92.2 16.9A 146.3 25.3B 60.0 26.8 64.6 18.9 20.9 4.6 23.7 5.4 120.0 48.3a 199.8 39.3b 10.1 3.2 10.9 2.8 9.2 3.6 15.8 6.5 UD
186.1 54.3 260.0 75.7
188.4 57.5 251.0 57.3
a,b
P < 0.05, A,BP < 0.01; UD e undetectable. SUM average of lactic, acetic, propionic and butyric acid concentration.
c
difference in the index of PA of monocytes, which was higher at day 14 (P < 0.05) in the BA group. The metabolic burst activity of granulocytes did not differ between the groups (P > 0.05, Table 5).
influenced significantly (Table 4) except for the concentration of haemoglobin, which was lower at days 14, 28 and 49 (P < 0.01) in the BA group. Eosinophil counts were decreased with significant difference at day 49 compared to control (P < 0.05).
4. Discussion 3.5. Phagocytic and metabolic burst activity of leukocytes 4.1. Characterization of B. animalis B/12 strain The total phagocytic activity (PA) of leukocytes was significantly higher (by over 10%) in the dogs after 14-day consumption of B. animalis B/12 (day 14, P < 0.01, Table 5); however, this was not so in the post-treatment period (day 28 and 49, P > 0.05). To see the results of cellular fraction of leukocytes, the PA of neutrophils was increased significantly (day 14, P < 0.01) compared to no visible differences in PA of monocytes (P > 0.05). There was significant
B. animalis B/12 was isolated from the faeces of a healthy dog (German Shepherd, female, 2 years old). In vitro, this strain was sensitive to most of the tested antimicrobial agents and showed inhibitory activity against Gram-negative bacteria (Enterobacter sp., E. coli, Klebsiella sp., Salmonella sp., Citrobacter sp.) based on production of organic acids [5]. B. animalis B/12 ferments 23/49 various
Table 3 Blood serum biochemical parameters (mean standard deviation) of the control group (C, n ¼ 10) and the group fed Bifidobacterium animalis B/12 (BA, n ¼ 10) for 14 days. Parameter
Group
Total protein (g/L)
C BA C BA C BA C BA C BA C BA C BA C BA C BA C BA C BA C BA
Day 0
Albumin (mmol/L) Urea (mmol/L) Triglyceride (mmol/L) Cholesterol (mmol/L) Glucose (mmol/L) Alanine aminotransferase (mkat/L) Aspartate aminotransferase (mkat/L) Alkaline Phosphatase (mkat/L) Glutathione peroxidase (mkat/L whole blood) Calcium (mmol/L) Phosphor (mmol/L) a,b
P < 0.05,
A,B
61.2 61.4 37.4 37.6 7.52 7.62 0.87 0.89 6.01 6.03 5.30 5.28 0.220 0.213 0.243 0.243 0.99 0.98 712 709 2.39 2.47 1.65 1.68
14
2.1 2.9 1.1 1.3 0.96 1.31 0.06 0.09a 0.98 1.28 0.27 0.31 0.088 0.048 0.046 0.068 0.36 0.27 69 76 0.18 0.16 0.07 0.12
61.2 61.0 37.6 34.9 7.47 7.23 0.88 0.82 5.98 5.77 5.28 5.12 0.203 0.329 0.251 0.383 1.01 1.38 718 758 2.41 2.40 1.63 1.61
28
2.8 3.6 1.5A 1.3B 1.14 1.00 0.06a 0.03b 1.14 1.00 0.17 0.19 0.030a 0.187b 0.095 0.178 0.21a 0.49b 81 106 0.13 0.20 0.10 0.09
61.0 61.1 37.3 35.6 7.37 7.38 0.87 0.84 5.72 5.68 5.30 5.20 0.210 0.214 0.246 0.270 1.01 1.45 710 701 2.39 2.40 1.64 1.64
49
1.9 2.8 1.1A 1.4B 1.02 1.13 0.06 0.04 1.03 1.30 0.24 0.36 0.068 0.063 0.061 0.058 0.25a 0.61b 65 81 0.26 0.16 0.09 0.12
61.3 62.0 37.5 36.3 7.49 7.80 0.87 0.83 6.03 6.05 5.28 5.28 0.206 0.259 0.241 0.246 1.06 1.30 714 758 2.38 2.37 1.63 1.65
2.1 2.0 1.1a 1.3b 0.93 0.77 0.07 0.04 0.78 1.24 0.16 0.27 0.096 0.130 0.088 0.042 0.58 0.46 87 123 0.11 0.16 0.14 0.09
P < 0.01.
Please cite this article in press as: Strompfová V, et al., Effect of Bifidobacterium animalis B/12 administration in healthy dogs, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.05.001
Q2
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Table 4 Haematological parameters (mean standard deviation) of the control group (C, n ¼ 10) and the group fed Bifidobacterium animalis B/12 (BA, n ¼ 10) for 14 days. Parameter
Group
Day 0
Red blood cells (G/L)
C BA C BA C BA C BA C BA C BA C BA C BA C BA
White blood cells (T/L) Haemoglobin (g/L) Haematocrit (L/L) Neutrophils (G/L) Lymphocytes (G/L) Monocytes (G/L) Eosinophils (G/L) Basophils (G/L) a,b
P < 0.05,
A,B
14
7.08 7.12 8.45 8.41 203.1 206.2 0.501 0.496 5.02 4.89 1.81 1.88 0.890 0.893 0.590 0.669 0.076 0.076
0.41 0.54 2.80 2.24 9.9 15.8 0.029 0.038 1.50 1.26 0.65 0.54 0.261 0.326 0.251 0.736 0.021 0.032
4.2. Faecal microflora and organic acid profile Consumption of B. animalis B/12 by dogs for 2 weeks led to a significant increase in LAB population (P < 0.05). The counts of bifidobacteria and enterococci were not influenced as well as the counts of not specified Clostridium-like bacteria. In contrast, the counts of coliforms were lower during and after treatment (P < 0.05). Almost no increase in faecal bifidobacteria is in accord with the canine study after consumption of B. animalis AHC7 for 6 weeks [9]. Despite the use of high doses of bifidobacteria (109 CFU) it seems that the adult canine intestinum at least is not a suitable environment for their colonization or multiplication (faecal population was up to 106 CFU/g). This opinion is also supported by the fact that bifidobacteria could not be isolated from all individual dogs [1,2]. Inhibitory effect of B. animalis B/12 against Gramnegative bacteria observed in vitro was also demonstrated in this
Table 5 Phagocytic activity and metabolic burst activity of leukocytes (mean standard deviation) measured in the control group (C, n ¼ 10) and in the group fed Bifidobacterium animalis B/12 (BA, n ¼ 10) for 14 days. Group
Day 0
FASUM FA Neu FA Mo IFASUM IFA Neu IFA Mo SI
0.59 0.64 2.10 2.37 10.0A 13.7B 0.031 0.037 2.11 1.82 0.50 0.68 0.301 0.382 0.462 0.425 0.044 0.063
7.10 7.29 8.57 9.04 203.9 187.7 0.506 0.508 5.60 6.06 1.78 1.68 0.810 0.810 0.507 0.451 0.067 0.072
49
0.39 0.68 1.90 2.92 12.5A 11.9B 0.041 0.043 2.08 3.05 0.62 0.35 0.296 0.294 0.370 0.543 0.032 0.047
7.12 7.16 8.60 9.62 199.3 182.1 0.502 0.498 5.20 6.60 1.73 1.92 0.834 0.856 0.430 0.162 0.073 0.086
0.55 0.71 2.90 2.70 8.9A 14.8B 0.039 0.044 1.99 2.29 0.49 0.50 0.670 0.361 0.300a 0.141b 0.026 0.038
P < 0.01.
carbohydrates with moderate b-galactosidase activity and survives in the presence of 0.3% bile and in artificial gastric juice (pH 2.5) sufficiently (decrease only by 0.5 log10 CFU/mL after 180 min, [5]).
Parameter
7.10 7.34 8.60 9.33 201.1 184.1 0.499 0.511 5.38 6.11 1.62 1.79 0.810 0.771 0.530 0.477 0.076 0.074
28
C BA C BA C BA C BA C BA C BA C BA
64.8 65.7 61.0 61.2 90.1 88.8 5043 5007 4280 4389 7111 7228 40.6 41.1
14
2.20 3.4 1.9 2.8 4.8 8.2 980 1359 655 972 810 1355 9.4 15.3
65.2 78.5 62.5 72.9 89.8 92.3 5001 5933 4870 5100 7200 8173 40.1 36.1
28
A
3.0 2.1B 2.3A 1.9B 2.8 3.6 1340 681 455 488 643a 915b 8.8 11.1
65.0 62.7 61.4 57.8 90.2 89.0 5021 5450 4970 4822 7577 7452 39.0 29.9
49
3.1 7.4 3.0 7.1 3.0 5.0 450 778 321 685 932 1203 12.0 10.8
63.7 65.7 62.9 64.9 90.2 90.4 4989 4764 4823 4643 7122 6640 39.0 41.3
2.4 6.2 2.8 7.4 1.9 2.4 541 882 630 821 805 807 5.1 6.9
FA e phagocytic activity; IFA e index of phagocytic activity; SI e stimulation index; Neu e neutrophils; Mo e monocytes. a,b P < 0.05, A,BP < 0.01.
in vivo study. The faecal coliforms were reduced from the end of the application period (day 14) till the end of the experiment (5 weeks after cessation of supplementation). Concurrently, significantly higher concentrations of acetic, valeric and acetoacetic acid were detected in the faeces. However, antimicrobial activity of lactobacilli and bifidobacteria against Gram-negative pathogenic bacteria is well known [10], although the mode of action of organic acid in the animal digestive tract is not fully understood. It has been assumed that undissociated forms of organic acids penetrate the lipid membrane of the bacterial cell and dissociate within the cell. As bacteria maintain the neutral pH of the cytoplasm, the export of excess protons consumes cellular ATP and results in depletion of energy [11]. The major end product of carbohydrate metabolism in bifidobacteria is acetate (B. animalis B/12 lactate:acetate 1:2.6 in vitro) which has higher pKa in comparison with lactic acid and therefore higher toxicity [12]. Since the proportion of undissociated molecules is dependent on pH values, the mean pH value during the treatment period was low enough (5.7, day 7 and 14) to exert a dissociative effect. A further mechanism of bacterial growth inhibition by acetic acids was proposed currently through genes expression control (involved in cellular metabolism and in the antiinflammatory response) or through the prevention of transepithelial electrical resistance reduction of the colonic epithelium, thereby improving intestinal defence [13]. The antimicrobial spectrum of acetate is very broad and in some cases more effective than common antiseptic solutions, e.g. against Pseudomonas aeruginosa [14]. Concerning the changes in acid concentrations, different results were obtained after application of Lactobacillus fermentum CCM 7421 to dogs, when butyric, succinic, formic and valeric acids were increased significantly [15]. 4.3. Faecal characteristics In our experiment, the faecal pH was decreased only by 0.2 (from 5.9 to 5.7, P > 0.05). It is similar to a value found in the canine experiment after supplementation with L. fermentum CCM 7421 [15]. Faecal consistency was visibly softer after the administration of B. animalis B/12 (increase by 0.3, P > 0.05); however, dry matter was almost not changed (difference up to 0.7%; P > 0.05). Sunvold et al. reported that faecal consistency score is more indicative of faecal characteristics than dry matter (poor correlations between faecal dry matter and faecal consistency exist [16]). Whereas the
Please cite this article in press as: Strompfová V, et al., Effect of Bifidobacterium animalis B/12 administration in healthy dogs, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.05.001
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addition of beneficial LAB to dogs with diarrhoea resulted in more solid faecal consistency [17], it seems the addition of the same doses of probiotic bacteria to healthy dogs (with balanced intestinal microbiota) leads vice versa to more liquid faeces. 4.4. Blood biochemistry In blood serum, decrease in albumin (P < 0.05) and triglyceride (P < 0.05) concentrations and increase in ALT (P < 0.05) and ALP (P < 0.05) was observed compared with control dogs. The concentration of albumin fraction decreased significantly from day 14 till the end of the study (by up to 2.7 g/L); however, the total protein content was not changed. Albumin, a measure of nutritional status, is the most osmotically active plasma protein responsible for about 75% of colloid oncotic pressure. A major metabolic function of albumin is general binding and transport [18]. Inflammation and malnutrition reduce albumin concentrations by decreasing its rate of synthesis and accelerating its degradation [19]. To outline the link between albumin and diarrhoea occurrence, every 1 g/L increase in albumin concentration was associated with an 18% reduction in the odds of C. difficile associated diarrhoea [20]. Despite a decrease of albumin in our case, no dog had an albumin level below 33 g/L during the experiment, and thus no hypoalbuminaemia was observed. These results are contrary to our previous experiments using L. fermentum CCM 7421, where elevation by 1.5 g/L was detected [15]. Bifidobacteria or other LAB are often studied for lipid-lowering effects and reduction of body weight in human or mice experiments [21]. In this canine experiment, the mean body weight of the dogs was almost without change (decrease by 0.3 kg, day 14, P > 0.05). The concentration of triglyceride was lower at the end of treatment (P < 0.05), whereby cholesterol level was not reduced despite an increase of acetate concentrations, the primary substrate for cholesterol synthesis, [22]. On the other hand, some short-chain fatty acids (SCFA, e.g. propionate) decrease lipid concentrations by reducing acetate utilization for fatty acid and cholesterol synthesis in the liver. Since 90e95% of SCFA are rapidly absorbed in the large intestine the real proportion of SCFA absorbed into the blood does not reflect faecal concentrations [23]. Concerning serum transaminases, some elevation of both tested enzymes (ALT, AST) was detected during the treatment, followed by a decrease in the post-application period. One dog had the activity of AST and ALT over 0.7 at day 14, but this had decreased by the next sampling (day 28, 49). The available evidence suggests that some probiotic strains including Bifidobacterium sp. are capable of preventing gut permeability, bacterial translocation, endotoxemia (associated with reduction of hepatic enzyme activity) and pro-inflammatory cytokine production in liver injury models [24]. In our case, liver activity was increased, most probably by metabolically active SCFA; it seems, this effect also depends on the health status of the animal. Most studies including experiments with the application of L. fermentum CCM 7421 to dogs have demonstrated reduction in hepatic enzyme activity in individuals with liver damage (e.g. inflammatory bowel disease, alcohol-induced liver injury, [17,24]. The activity of ALP was also significantly higher from day 14 compared to control, and could indicate intestinal or tissue (liver, bones, kidneys) activity or damage. Reinforced activity of bone tissue cells could balance the acidifying effect of resorbed SCFA (to retain optimal serum pH and mineral levels). 4.5. Haematology and phagocytic activity of leukocytes In this study, significant decrease in haemoglobin concentration (P < 0.01, within the limits of the physiological range) was noted at day 14 and 2e5 weeks after cessation of supplementation; however,
the red blood cell count was not changed. The decrease in haemoglobin could again be connected with the fermentation metabolites (mostly SCFA) and the diet composition (a larger amount of carbohydrates produces a larger amount of SCFA). Recently, increasing evidence for haemoglobin reducing effect caused by LAB exists [25,26]; therefore, it is necessary to test haematological parameters which have often been omitted in animal studies. Concerning the effect of B. animalis B/12 on the activation of blood macrophages, the total PA of leukocytes (P < 0.01) and PA of neutrophils (P < 0.01) were significantly higher during the supplementation phase (day 14) compared to control. The index of PA was increased significantly only in monocytes (P < 0.05). Significant stimulation of PA in granulocytes is consistent with our previous canine study [15]; however, the time of PA increase and the length of this increase differed (the same experimental design was used). Whereas PA increased progressively after application of L. fermentum CCM 7421 with the significant difference in the post-treatment period (day 49), B. animalis B/12 resulted in immediate increase of PA during the treatment (day 14) with decrease to basal values in the post-treatment period. While phagocytosis increased, respiratory burst activity tended to decrease (day 14, 28) with no significant difference. Although the exact mode of stimulation is not well understood, phagocytic cells are probably stimulated after direct contact with LAB or sub-cellular fractions of bacterial cell walls. They can induce mononuclear leukocytes to secrete procellular immunity cytokines, which serves to drive the immune system towards a general enhancement of cellular immune function [27]. In conclusion, this study demonstrates that application of B. animalis B/12 to healthy dogs is associated with the reduction of coliform bacteria in faeces, decrease in haemoglobin, albumin and triglyceride serum concentrations and increase in ALT, ALP and PA of neutrophils. It seems that daily long-term applications of LAB in higher doses (over 108 CFU) to healthy dogs are not desirable. According to presented and our previous results, many of the effects lasted for several weeks after the treatment period. This means that some of the LAB effects are persistent in the long term. Acknowledgements The study was financially supported by the Slovak Scientific Agency VEGA, project no. 2/0056/13. The help of dogs owners is gratefully acknowledged. References [1] Greetham HL, Giffard C, Hutson RA, Collins MD, Gibson GR. Bacteriology of the Labrador dog gut: a cultural and genotypic approach. J Appl Microbiol 2002;93:640e6. [2] Handl S, Dowd SE, Garzia-Mazcorro JF, Steiner JM, Suchodolski JS. Massive parallel 16S rRNA gene pyrosequencing reveals highly diverse fecal bacterial and fungal communities in healthy dogs and cats. FEMS Microbiol Ecol 2011;76:301e10. [3] Kim SY, Adachi Y. Biological and genetic classification of canine intestinal lactic acid bacteria and bifidobacteria. Microbiol Immunol 2007;51:919e28. Svobodová I, Jebavý L, et al. Bifi[4] Bunesová V, Vlková E, Rada V, Ro cková S, dobacterium animalis subsp. lactis strains isolated from dog faeces. Vet Microbiol 2012;160:501e5. [5] Strompfová V, Lauková A. Isolation and characterization of faecal bifidobacteria and lactobacilli isolated from dogs and primates. Anaerobe; 2014. http:// dx.doi.org/10.1016/j.anaerobe.2013.10.007. [6] Lee J-H, O’Sullivan DJ. Genomic insights into bifidobacteria. Microbiol Mol Biol Rev 2010;74:378e416. [7] Picard C, Fioramonti J, Francois A, Robinson T, Neant F, Matuchansky C. Review article: bifidobacteria as probiotic agents e physiological effects and clinical benefits. Aliment Pharmacol Ther 2005;22:495e512. [8] Kelley R, Levy K, Mundell P, Hayek MG. Effects of varying doses of a probiotic supplement fed to healthy dogs undergoing kenneling stress. Int J Appl Res Vet Med 2012;10:205e16. [9] ÓMahony D, Barry Murphy K, MacSharry J, Boileau T, Sunvold G, Reinhart G, et al. Portrait of a canine probiotic Bifidobacterium e from gut to gut. Vet Microbiol 2009;139:106e12.
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[19] Don BR, Kaysen G. Serum albumin: relationship to inflammation and nutrition. Semin Dial 2004;17:432e7. [20] Hickson M, D’Souza AL, Muthu N, Rogers TR, Want S, Rajmkumar C, et al. Use of probiotic Lactobacillus preparation to prevent diarrhoea associated with antibiotics: randomised double blind placebo controlled trial. BMJ 2007;335: 80. [21] Cano PG, Santacruz A, Trejo FM, Sanz Y. Bifidobacterium CECT 7765 improves metabolic and immunological alterations associated with obesity in high-fat diet-fed mice. Obesity 2013;21:2310e21. [22] Pereira DIA, Gibson GR. Effects of consumption of probiotics and prebiotics on serum lipid levels in humans. Crit Rev Biochem Mol Biol 2002;37:259e81. [23] Hijova E, Chmelarova A. Short chain fatty acids and colonic health. Bratisl-Lek Listy 2007;108:354e8. [24] Kirpich IA, Solovieva NV, Leikhter SN, Shidakova NA, Lebedeva OV, Sidorov PI, et al. Probiotics restore bowel flora and improve liver enzymes in human alcohol-induced liver injury: a pilot study. Alcohol 2008;42:675e82. [25] Uchida M, Tsuboi H, Takahashi M, Nemoto A, Seki K, Tsunoo H, et al. Safety of high doses of Propionibacterium freundenreichii ET-3 culture in healthy adult subjects. Reg Toxicol Pharmacol 2011;60:262e7. [26] Dahyia T, Sihag RC, Gahlawat SK. Effect of probiotics on the haematological parameters of Indian Magur (Clarius batrachus L.). J Fisch Aquat Sci; 2012: 279e90. [27] Chiang BL, Sheih YH, Wang LH, Liao CK, Gill HS. Enhancing immunity by dietary consumption of a probiotic lactic acid bacterium (Bifidobacterium lactis HN019): optimization and definition of cellular immune responses. Eur J Clin Nutr 2000;54:849e55.
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