Journal of Equine Science. Dec/31/2013; 25(2): 37-43

Published online Jun/24/2014

PMID: 25013357

PMC: 4090357

doi: 10.1294/jes.25.37

Anti-inflammatory and Intestinal Barrier–protective Activities of Commensal Lactobacilli and Bifidobacteria inThoroughbreds: Role of Probiotics in Diarrhea Prevention in Neonatal Thoroughbreds

Kiyoshi SUGAYA+6 authors

ABSTRACT

We previously isolated the commensal bacteria lactobacilli and bifidobacteria from the Thoroughbred intestine and prepared thehorse probiotics LacFi^TM, consisting of Lactobacillus ruminis KK14, L. equi KK 15, L. reuteri KK18, L. johnsonii KK21,and Bifidobacterium boum HU. Here, we found that the five LacFi^TM constituent strains remarkably suppressedpro-inflammatory interleukin-17 production in mouse splenocytes stimulated with interleukin-6 and transforming growth factor-β.The protective effects of the probiotic on impaired intestinal barrier function were evaluated in Caco-2 cells treated with tumornecrosis factor-α. Evaluation of transepithelial resistance showed that all the strains exhibited intestinal barrier protectiveactivity, with significant suppression of barrier impairment by L. reuteri KK18. The LacFi^TM constituent strains weredetected in neonatal LacFi^TM-administered Thoroughbred feces using polymerase chain reaction denaturing gradient gelelectrophoresis and culture methods. These five strains were found to be the predominant lactobacilli and bifidobacteria in theintestinal microbiota of LacFi^TM-administered Thoroughbreds. Administration of LacFi^TM to neonatalThoroughbreds decreased diarrhea incidence from 75.9% in the control group (n=29 neonatal Thoroughbreds) to 30.7% in theLacFi^TM-administered group (n=101 neonatal Thoroughbreds) immediately after birth to 20 weeks after birth.LacFi^TM treatment also prevented diarrhea especially at and around 4 weeks and from 10 to 16 weeks. The duration ofdiarrhea was also shorter in the probiotics-administered group (7.4 ± 0.8 days) than in the control group (14.0 ± 3.2 days). Theseresults indicate that the LacFi^TM probiotics regulates intestinal function and contributes to diarrhea prevention.

The incidence of diarrhea in Thoroughbred yearlings is very high, sometimes resulting in severe maldevelopment and even death. Asphysical maldevelopment and mental stress may negatively affect Thoroughbred performance, the avoidance of diarrhea in Thoroughbredyearlings is important for breeders and veterinarians.

Lactobacilli and bifidobacteria are important constituents of the healthy gastrointestinal tract of mammals and humans, and somestrains of lactobacilli and bifidobacteria are frequently administered as probiotics because of their beneficial roles in mammal andhuman health and diarrhea prevention [1, 5, 6, 19]. The existence of lactobacilli in the intestinal floraof Thoroughbreds has long been suggested [9]. Morotomi et al. [15], Endo et al. [4], and Morita etal. [13, 14] isolated lactobacilli from horsefecal samples, including Thoroughbreds. We also isolated Bifidobacterium boum from the intestinal flora ofThoroughbreds [3]. Based on the above studies, a horse probiotics consisting ofLactobacillus ruminis KK14, L. equi KK15, L. reuteri KK18, L.johnsonii KK21 [13], and B. boum HU [4] was prepared and termed LacFi^TM. LacFi^TM has been recognized as “Generally Recognized As Safe(GRAS)” following use for >11 years.

It has recently been reported that some probiotic strains reduce the incidence of antibiotics and Clostridiumdifficile-associated diarrhea [7, 26]. Thesebacteria have been shown to increase beneficial intestinal bacteria and to modulate immune function. Several intestinal toxins induceepithelial barrier dysfunction through changes in tight junction (TJ) proteins that contribute to diarrhea [17]. We reported that Enterococcus hirae, an intestinal bacterium in the adjacent mucosa (mucosalbacterium), ameliorates tumor necrosis factor (TNF)-α-induced barrier impairment in the human epithelial TJ [10]. Therefore, immunomodulation, such as suppression of inflammation, and regulation of TJ function are beneficialfor preventing diarrhea.

In this study, we investigated the regulative effects of the five LacFi^TM constituent bacterial strains (four lactobacilliand one bifidobacterium) on splenocytes and intestinal epithelial cells to evaluate the effect of the probiotics on diarrheaprevention. The LacFi^TM constituent strains were detected in neonatal Thoroughbred feces by polymerase chain reactiondenaturing gradient gel electrophoresis (PCR-DGGE) and culture methods. We also evaluated the incidence of diarrhea in neonatalThoroughbreds after the administration of LacFi^TM, with a focus on the anti-inflammatory and the intestinalbarrier–protective activities.

Materials and Methods

Reagents

RPMI 1640 and Dulbecco’s modified Eagle’s medium, nonessential amino acids, penicillin, streptomycin, and gentamycin were allobtained from Life Technologies (Forster City, CA, USA). Fetal bovine serum (FBS) was obtained from MP Biomedicals, (Osaka,Japan). Both recombinant human transforming growth factor (TGF)-β and mouse interleukin (IL)-6 were obtained from R&D Systems(Minneapolis, MN, USA). TNF-α was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). MRS broth and MRS agar wereobtained from Becton, Dickinson and Company (Sparks, MD, USA). All other chemicals were of reagent grade.

Bacterial culture preparation for studies on anti-inflammatory and intestinal barrier-protective activities

Bacteria were cultured in MRS broth and incubated under anaerobic conditions by using AnaeroPack^TM (Mitsubishi GasChemical Company, Inc., Tokyo, Japan) at 37°C for 24 hr. The cell suspensions were washed with distilled water, incubated at 100°Cfor 50 min [10], and then lyophilized. Subsequently, the heat-killed bacteria were added tomurine splenocyte or Caco-2 cell culture.

Splenocyte culture

The suppressive effects of bacteria on IL-17 production were evaluated according to previously described methods [18, 23]. Briefly, 6-week-old female Balb/c mice wereobtained from Charles River Laboratories International, Inc. (Kanagawa, Japan) and killed by cervical dislocation. Spleens wereremoved from three mice for each experiment and the pooled splenocytes (10⁷ cells) were incubated with TGF-β (2ng/ml) and IL-6 (20 ng/ml) at 37°C for 72 hr in 1 ml of RPMI 1640 mediumsupplemented with 10% FBS, 10 μM 2-mercaptoethanol, 10 mM HEPES, penicillin, and streptomycin. Heat-killedbacteria (10⁷ cells) were added to the culture. A culture without the addition of TGF-β, IL-6, or heat-killed bacteriawas included as a control. Culture supernatants were harvested to measure IL-17 concentrations by sandwich enzyme-linkedimmunosorbent assay (ELISA) (R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions.

Caco-2 cell culture

Caco-2 cells were purchased from the American Type Culture Collection (Rockville, MD, USA). In this study, Caco-2 cells were usedbetween passages 35 and 45. The growth medium consisted of Dulbecco’s modified Eagle’s medium with 10% FBS, 1% nonessential aminoacids, and antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin, and 50μg/ml gentamycin). Cells were cultured at 37°C under a humidified 5% CO₂ atmospherein 75-cm² tissue culture flasks to approximately 80% confluence and seeded into a 12-well Transwell cell culturechamber (0.4-μm pore size, 12-mm diameter) (Corning Inc., Tewksbury, MA, USA) at a density of 5 × 10⁴cells/cm². After 14 days of culture, transepithelial resistance (TER) was measured using a Millicell-ERS instrumentwith Ag/AgCl electrodes (EMD Millipore Corporation, Billerica, MA, USA). Caco-2 cell monolayers were used when their TER valueswere >300 ohm•cm². Each well was placed in a cluster plate with an external medium (basolateral side, 1.5ml) and an internal medium (apical side, 0.5 ml). The cell monolayers were fed fresh mediumevery 24 hr.

Evaluation of epithelial barrier function

The protective effects of bacteria on epithelial impairment were evaluated according to previously described methods [10, 11]. Briefly, bacteria (10⁵ cells/well)were added to the apical side of the Caco-2 cell monolayers. One hour later, the cells were treated with TNF-α (100ng/ml) on the basolateral side of the cell monolayers and cultured for 48 hr. After the 48-hr incubation, theTER value was measured to assess epithelial barrier function.

Bacterial culture preparation for administration to neonatal Thoroughbreds

L. ruminis KK14, L. equi KK15, L. reuteri KK18, L. johnsoniiKK21 [13], and B. boum HU [4]strains produced by Crossfield-Bio, Inc. (Tokyo, Japan) were combined to prepare the probiotic LacFi^TM in this study.Using 10 Sprague-Dawley rats and 10 Balb/c mice (Charles River Laboratories International, Inc.), these bacterial strains(10¹⁰ colony forming units (CFU)/head) had been previously evaluated to elicit no sign of illness and toxicity (suchas full blood count, hematocrit, mean corpuscular volume [MCV], erythrocyte sedimentation rate [ESR], aspartate transaminase[AST], alkaline phosphatase [ALP], lactate dehydrogenase [LDH], glutamic oxaloacetic transaminase [GOT], and glutamic pyruvatetransaminase [GPT]) over 6 months by veterinarians. All animal experiments were performed in accordance with guidelines for thecare and use of laboratory animals. Each strain was cultured separately in MRS broth (Kanto Chemical Co., Inc., Tokyo, Japan) andincubated at 37°C for 24 hr. Bacterial cells were washed twice with saline by centrifugation. The cell pellets were resuspendedwith sterilized 10% skim milk to the final concentration of CFU/ml shown in Table 1

Table 1.

LacFi^TM-constituent strains and cell counts before/after freezing at –80°C

Species	Strain	Cell counts before freezing (CFU/ml)	Cell counts after freezing (CFU/ml)
Lactobacillus reuteri	KK18	2.0 × 10⁹	1.6 × 10⁹
Lactobacillus ruminis	KK14	1.2 × 10⁹	0.9 × 10⁹
Lactobacillus equi	KK15	5.8 × 10⁹	4.8 × 10⁹
Lactobacillus johnsonii	KK21	6.4 × 10⁹	5.9 × 10⁹
Bifidobacterium boum	HU	7.9 × 10⁹	6.1 × 10⁹
Mixture of the above five strains*		9.8 × 10⁹	8.6 × 10⁹

* The mixture was administered to neonatal Thoroughbreds as LacFi^TM in this study.

and stored at −80°C until administration. Each thawed suspension contained more than 10⁹CFU/ml of living bacteria.

Neonatal Thoroughbreds: Observation of animals and clinical evaluation

One hundred and thirty healthy neonatal Thoroughbreds at ages ranging from immediately after birth to 20 weeks of age wereincluded in the study from January to May 2008. This evaluation was carried out with sufficient attention given to animalprotection. The neonatal Thoroughbreds belonged to the research herd at Northern Farm in Hokkaido, Japan. Prior to studycommencement, the neonatal Thoroughbreds had been determined to have no signs of illness by veterinarians. TheLacFi^TM-administered group (n=101) was administered 50 ml of probiotics in skim milk, and the controlgroup (n=29) was untreated. Following study commencement, the neonatal Thoroughbreds were evaluated for signs of illness anddiarrhea every 2 weeks by veterinarians for up to 20 weeks after birth. In this study, although we did not evaluate subjects withthe blind study, we evaluated them with the random control group study.

Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE)–based detection of lactobacilli

Meconium and feces were collected on the 7th, 14th, and 21st days after birth from five neonatal Thoroughbreds of both thecontrol and LacFi^TM-administered groups. DNA was extracted from the fecal samples and PCR-DGGE analysis was performedusing a previously described method [12, 16].Approximately 340 bp of the 16S rRNA gene of Escherichia coli No. 341–534 were amplified by PCR using the Lac1and Lac2GC primer sets [25]. The primer set used for PCR-DGGE comprised the forward primerLac1 (5′-AGCAGTAGGGAATCTTCCA-3′) and the reverse primer Lac2GC (5′-CGCCCGGGGCGCGCCCCGGGCGGCCCGGGGGCACCGGGGGATTYCACCGCTACACATG-3′).The PCR fragments were separated by DGGE using the DCode system (Bio-Rad Laboratories, Hercules, USA) with the followingmodifications: For the DGGE analysis of bacteria, 8% (wt/vol) polyacrylamide gels were prepared using a denaturing gradientranging from 35 to 50%, and electrophoresis was performed in Tris-acetate-EDTA (TAE) buffer for 14 hr at a constant voltage of 60V. A 100% denaturant corresponded to 7 M urea and 40% formamide. After electrophoresis, the gels were stained for 15 min using anethidium bromide solution (250 ml TAE buffer + 25 µl of 10 mg/ml ethidiumbromide solution). The PCR products derived from the DGGE bands were purified with the Wizard SV Gel and PCR Clean-up system(Promega Co., Madison, USA). The primers used for sequencing were the same as those used for the re-amplification of DNAs fromDGGE bands. Sequence similarities between the DNAs were determined using Standard Nucleotide BLAST (BLASTN)(http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Statistical analyses

Differences between measured values were analyzed using Tukey-Kramer’s test, Student’s t-test and Fisher’s exacttest, and P-values <0.05 were considered statistically significant.

Results and Discussion

The pathogenesis of diarrhea, including the effects of physiological and mental stress, in Thoroughbred yearlings is complex, andthe resulting intestinal inflammation is responsible for the occurrence of diarrhea. The anti-inflammatory activity of the fiveLacFi^TM constituent strains, L. ruminis KK14, L. equi KK 15, L.reuteri KK18, L. johnsonii KK21, and B. boum HU, was examined invitro. Elevated levels of pro-inflammatory cytokines such as IL-17 and chemokines, a subset of chemoattractant cytokines,are observed in the inflamed intestine and are critically involved in the pathogenesis of intestinal inflammation [8, 22]. In this study, IL-17 production by mousesplenocytes was evaluated. The addition of TGF-β and IL-6 drastically enhanced IL-17 production by splenocytes (Fig. 1

Fig. 1.

Suppressive effects of LacFi^TM constituent bacteria on pro-inflammatory interleukin (IL)-17 production in murinesplenocytes. Splenocytes were stimulated with TGF-β and IL-6. Bacteria were added to the media and cultured for 72 hr. Culturesupernatants were harvested and assayed for IL-17 concentrations. The culture alone (Cont) and culture without the addition ofbacteria (–) were included as controls. Results are expressed as mean ± SD values (n=3). **P<0.01 comparedto control (–) (Tukey-Kramer).

); however, all five strains in LacFi^TM significantly (P<0.01) suppressed the production ofIL-17. Among the five strains, L. reuteri KK18 suppressed IL-17 production most potently. We have reported thesimilar suppressive effects of Streptococcus thermophilus ST28 and its genomic DNA fraction on Th17 response inmurine splenocytes stimulated with TGF-β plus IL-6 [24]. Several studies showed thatToll-like receptor (TLR) 9-signalling had an important role for modulating experimental allergic encephalomyelitis and colitis. Forexample, it was reported that TLR9 ligands mediated the anti-inflammatory effects of probiotic bacteria in murine experimentalcolitis [20]. Therefore, it was likely that genomic DNA of the LacFi^TM constituentstrains suppressed IL-17 production, at least in part, via TLR9.

Next, the protective effects of the LacFi^TM constituent strains on epithelial barrier impairment were evaluated, sinceintestinal barrier impairment is also involved in diarrhea. TNF-α is an essential mediator of inflammation in the gut. Severalpro-inflammatory actions have been proposed for TNF-α, such as the production and stimulation of pro-inflammatory cytokines andactivation of the acute-phase response. TNF-α induces an increase in intestinal TJ permeability [2]. In this study, TJ barrier impairment was induced by TNF-α in the human epithelial Caco-2 cells, and the protectiveeffects of LacFi^TM constituent bacteria on the impairment were evaluated by measuring the TER value, reflecting TJpermeability. As shown in Fig. 2

Fig. 2.

Protective effects of LacFi^TM constituent bacteria with respect to intestinal barrier impairment. Caco-2 cellswere treated with the bacterium or medium alone (–) for 1 hr and exposed to tumor necrosis factor-α for 48 hr. Afterincubation, the transepithelial resistance value was measured to assess intestinal barrier function. The culture alone (Cont)and culture without the addition of bacteria (–) were included as controls. Results are expressed as the relative values andmean ± SD values (n=3). *P<0.05 compared to control (–) (Tukey-Kramer).

, TNF-α decreased the TER value by approximately 25%; however, pretreatment with L. reuteri KK18significantly (P<0.05) suppressed the TNF-α-induced decrease in TER. L. johnsonii KK21 alsosuppressed the decrease in TER to some extent. We have reported that Enterococcus hirae ATCC 9790^T andits cell wall fractions protected against intestinal impairment by regulation of epithelial tight junction via TLR2 signaling. Inaddition, Cario et al. [3] reported thatPam₃Cys-Ser-(Lys)₄, a TLR2 ligand, ameliorated colonic inflammation in mice with colitis induced by dextransodium sulfate. It was probable that cell wall fractions of the LacFi^TM constituent strains suppressed the intestinalbarrier impairment in the present study, although further investigations are necessary to clarify this.

A defective intestinal epithelial TJ barrier characterized by an increase in intestinal permeability is an important pathogenicfactor contributing to the development of intestinal inflammation resulting in diarrhea. Animal studies have shown that enhancementof the intestinal TJ barrier prevents cytokine-mediated development of intestinal inflammation and diarrhea [2]. Clinical studies have also shown that anti–TNF-α therapy recovers the intestinal barrier and thatnormalization of intestinal permeability is associated with long-term clinical remission [21]. In this regard, it was hypothesized that LacFi^TM constituent probiotics would prevent diarrhea throughmodulation of the immune response and improvement in TJ function.

Considering the interesting results obtained in vitro, we evaluated the in vivo effect ofLacFi^TM constituent probiotics on the incidence of diarrhea in neonatal Thoroughbreds after the administration ofLacFi^TM. The frozen bacterial mixture LacFi^TM was administered to neonatal Thoroughbreds orally at a dose of8.6 × 10⁹ CFU/50 ml (Table 1) on the 2nd, 3rd, 4th, and 5thdays after birth; LacFi^TM administration was then continued with one administration/week up to 4 weeks. After the 20-weekexperimental period, oral administration of LacFi^TM to 101 neonatal Thoroughbreds did not result in reduced body weight.Also, no signs of toxicity were observed in any animal and no animals died during the test period.

PCR-DGGE analysis confirmed that L. ruminis, L. equi, L. reuteri, L.johnsonii, possibly derived from LacFi^TM, colonized the neonatal Thoroughbred intestine immediately afterbirth under germ-free conditions (Fig. 3-A

Fig. 3.

DGGE analysis of the PCR products of lactic acid bacteria present in the neonatal Thoroughbred feces by lactic acidbacteria-specific primers [25]. Approximately 200 bp 16S rDNA of E.coli No. 341–534 were amplified by PCR. Lane 1: meconium, lane 2: feces obtained on the 7th day after birth, lane3: feces obtained on the 14th day after birth, lane 4: feces obtained on the 21st day after birth. Band a: L.johnsonii (100% similarity), band b: L. equi (100% similarity), band c: L.ruminis (99.9% similarity), and band d: L. reuteri (99.8% similarity).

). The broad band (Fig. 3-B) corresponding to the LacFi^TM species was notdetected in the analysis of the control group (LacFi^TM not administered) fecal DNA.

B. boum was isolated from the feces of LacFi^TM-administered neonatal Thoroughbreds by culture methodsusing MRS agar under anaerobic conditions at 37°C for 48 hr but not in the feces of the control group. As bifidobacteria comprise aminority of the fecal microbiota of the Thoroughbred [3], it is likely that theBifidobacterium isolated from the intestinal tract originated from the probiotics.

The symptom of diarrhea was also compared between the LacFi^TM-administered and control groups. The Thoroughbreds wereevaluated for diarrhea every 2 weeks, and the ratio (%) of horses which were suffered from diarrhea at 2, 4, 6, 8, 10, 12, 14, 16,18 and 20 weeks were shown in Fig. 4

Fig. 4.

Comparison of the number of neonatal Thoroughbreds showed the symptom of diarrhea in the LacFi^TM-administered andcontrol groups. The ratio (%) of horses which were suffered from diarrhea at 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 weeks wereshown.

. Administration of LacFi^TM to the neonatal Thoroughbreds decreased the incidence of diarrhea from 75.9% in thecontrol group (n=29 neonatal Thoroughbreds) to 30.7% in the LacFi^TM-administered group (n=101 neonatal Thoroughbreds).The control group, which was not administered LacFi^TM, exhibited diarrhea after birth up to 2 weeks, and theLacFi^TM-treated group had markedly lesser diarrhea at all these timepoints, especially at and around 4 weeks and from10 to 16 weeks (Fig. 4). The duration of diarrhea was also shorter in theprobiotics-administered group (7.4 ± 0.8 days) than in the control group (14.0 ± 3.2 days) (Table 2

Table 2.

Comparison of clinical manifestations of diarrhea in the LacFi^TM-administered and control groups

	LacFi^TM-administered	Not administered (control)
Total number of neonatal Thoroughbreds	101	29
Number of neonatal Thoroughbreds exhibiting diarrhea *	31	22
Ratio of diarrhea in neonatal Thoroughbreds (%)	30.7	75.9
Duration of diarrhea (days) (mean ± standard error of the mean) **	7.4 ± 0.8	14.0 ± 3.2

* Number of neonatal Thoroughbreds exhibiting diarrhea in LacFi^TM-administered group was significantly(P<0.01) lower than that in control group, judged by Fisher’s exact test. ** Duration of diarrhea ofLacFi^TM-administered group was significantly (P<0.001) shorter than that of control group,judged by Student’s t-test.

These results indicate that the horse probiotic LacFi^TM regulates intestinal function and contributes to diarrheaprevention. LacFi^TM treatment may be useful even if diarrhea develops, considering the impact of medical treatmentsadministered at this important time of Thoroughbred growth on future race performance.

The intestine not only digests and absorbs food but also functions as a part of the immune system critical for host-defense. Inaddition, the intestinal barrier acts as the first defense against a vast amount of food, exogenous antigens, and commensalbacteria; therefore, it is important to regulate the cytokine balance and maintain the intestinal barrier. Diarrhea prevention byLacFi^TM might be, at least in part, attributed to the improvement in cytokine balance and to the enforcement of the TJbarrier and subsequent maintenance of intestinal integrity [11, 18, 22,23,24].

References

1. AllenS.J.WarehamK.WangD.BradleyC.HutchingsH.HarrisW.DharA.BrownH.FodenA.GravenorM.B.MackD.2013Lactobacilli and bifidobacteria in the prevention of antibiotic-associated diarrhoea andClostridium difficile diarrhoea in older inpatients (PLACIDE): a randomised, double-blind,placebo-controlled, multicentre trial. Lancet382:1249–1257[PubMed][Google Scholar]
2. Al-SadiR.GuoS.YeD.MaT.Y.2013TNF-α modulation of intestinal epithelial tight junction barrier is regulated by ERK1/2activation of Elk-1. Am. J. Pathol.183:1871–1884[PubMed][Google Scholar]
3. CarioE.GerkenG.PodolskyD.K.2007Toll-like receptor 2 controls mucosal inflammation by regulating epithelial barrierfunction. Gastroenterology132:1359–1374[PubMed][Google Scholar]
4. EndoA.OkadaS.MoritaH.2007Molecular profiling of Lactobacillus, Streptococcus, andBifidobacterium species in feces of active racehorses. J. Gen. Appl.Microbiol.53: 191–200[PubMed][Google Scholar]
5. FukudaS.TohH.HaseK.OshimaK.NakanishiY.YoshimuraK.TobeT.ClarkeJ.M.ToppingD.L.SuzukiT.TaylorT.D.ItohK.KikuchiJ.MoritaH.HattoriM.OhnoH.2011Bifidobacteria can protect from enteropathogenic infection through production ofacetate. Nature469: 543–547[PubMed][Google Scholar]
6. HempelS.NewberryS.J.MaherA.R.WangZ.MilesJ.N.V.ShanmanR.JohnsenB.ShekelleP.G.2012Probiotics for the prevention and treatment of antibiotic-associated diarrhea: a systematicreview and meta-analysis. JAMA307:1959–1969[PubMed][Google Scholar]
7. HicksonM.D’SouzaA.L.MuthuN.RogersT.R.WantS.RajkumarC.BulpittC.J.2007Use of probiotic Lactobacillus preparation to prevent diarrhoea associated with antibiotics:randomised double blind placebo controlled trial. BMJ335:80[PubMed][Google Scholar]
8. KoelinkP.J.OverbeekS.A.BraberS.de KruijfP.FolkertsG.SmitM.J.KraneveldA.D.2012Targeting chemokine receptors in chronic inflammatory diseases: an extensivereview. Pharmacol. Ther.133:1–18[PubMed][Google Scholar]
9. MitsuokaT.KaneuchiC.1977Ecology of the bifidobacteria. Am. J. Clin.Nutr.30: 1799–1810[PubMed][Google Scholar]
10. MiyauchiE.MoritaH.OkudaJ.SashiharaT.ShimizuM.TanabeS.2008Cell wall fraction of Enterococcus hirae ameliorates TNF-alpha-induced barrierimpairment in the human epithelial tight junction. Lett. Appl.Microbiol. 46: 469–476[PubMed][Google Scholar]
11. MiyauchiE.MoritaH.TanabeS.2009Lactobacillus rhamnosus alleviates intestinal barrier dysfunction in part byincreasing expression of zonula occludens-1 and myosin light-chain kinase in vivo.J. Dairy Sci. 92: 2400–2408[PubMed][Google Scholar]
12. MoritaH.NakajimaF.MurakamiM.EndoA.SuzukiT.ShiratoriC.KatoY.OkataniT.A.AkitaH.MasaokaT.2007Molecular monitoring of the main changes in bacterial floral diversity in the gastrointestinaltract of a thoroughbred foal with catarrhal enteritis by using PCR-DGGE. J. Equine Vet.Sci. 27: 14–19[Google Scholar]
13. MoritaH.NakanoA.ShimazuM.TohH.NakajimaF.NagayamaM.HisamatsuS.KatoY.TakagiM.TakamiH.AkitaH.MatsumotoM.MasaokaT.MurakamiM.2009Lactobacillus hayakitensis, L. equigenerosi and L. equi,predominant lactobacilli in the intestinal flora of healthy thoroughbreds. Anim. Sci.J. 80: 339–346[PubMed][Google Scholar]
14. MoritaH.ShimazuM.ShionoH.TohH.NakajimaF.AkitaH.TakagiM.TakamiH.MurakamiM.MasaokaT.TanabeS.HattoriM.2010Lactobacillus equicursoris sp. nov., isolated from the faeces of a thoroughbredracehorse. Int. J. Syst. Evol. Microbiol. 60:109–112[PubMed][Google Scholar]
15. MorotomiM.YukiN.KadoY.KushiroA.ShimazakiT.WatanabeK.YuyamaT.2002Lactobacillus equi sp. nov., a predominant intestinal Lactobacillus species of the horseisolated from faeces of healthy horses. Int. J. Syst. Evol. Microbiol.52: 211–214[PubMed][Google Scholar]
16. MuyzerG.SmallaK.1998Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gelelectrophoresis (TGGE) in microbial ecology. Antonie vanLeeuwenhoek73: 127–141[PubMed][Google Scholar]
17. Ngendahayo MukizaC.DubreuilJ.D.2013Escherichia coli heat-stable toxin b impairs intestinal epithelial barrierfunction by altering tight junction proteins. Infect. Immun.81: 2819–2827[PubMed][Google Scholar]
18. OgitaT.NakashimaM.MoritaH.SaitoY.SuzukiT.TanabeS.2011Streptococcus thermophilus ST28 ameliorates colitis in mice partially bysuppression of inflammatory Th17 cells. J. Biomed. Biotechnol.2011: 378417[PubMed][Google Scholar]
19. OhlandC.L.MacnaughtonW.K.2010Probiotic bacteria and intestinal epithelial barrier function.Am. J. Physiol. Gastrointest. Liver Physiol.298:G807–G819[PubMed][Google Scholar]
20. RachmilewitzD.KatakuraK.KarmeliF.HayashiT.ReinusC.RudenskyB.AkiraS.TakedaK.LeeJ.TakabayashiK.RazE.2004Toll-like receptor 9 signaling mediates the anti-inflammatory effects of probiotics in murineexperimental colitis. Gastroenterology126:520–528[PubMed][Google Scholar]
21. SuenaertP.BulteelV.LemmensL.NomanM.GeypensB.Van AsscheG.GeboesK.CeuppensJ.L.RutgeertsP.2002Anti-tumor necrosis factor treatment restores the gut barrier in Crohn’sdisease. Am. J. Gastroenterol. 97:2000–2004[PubMed][Google Scholar]
22. TanabeS.2013The effect of probiotics and gut microbiota on Th17 cells. Int.Rev. Immunol. 32: 511–525[PubMed][Google Scholar]
23. TanabeS.KinutaY.SaitoY.2008Bifidobacterium infantis suppresses proinflammatory interleukin-17 productionin murine splenocytes and dextran sodium sulfate-induced intestinal inflammation. Int. J. Mol.Med. 22: 181–185[PubMed][Google Scholar]
24. OgitaT.TaniiY.MoritaH.SuzukiT.TanabeS.2011Suppression of Th17 response by Streptococcus thermophilus ST28 through induction ofIFN-γ. Int. J. Mol. Med. 28:817–822[PubMed][Google Scholar]
25. WalterJ.HertelC.TannockG.W.LisC.M.MunroK.HammesW.P.2001Detection of Lactobacillus, Pediococcus, Leuconostoc, andWeissella species in human feces by using group-specific PCR primers and denaturing gradient gelelectrophoresis. Appl. Environ. Microbiol. 67:2578–2585[PubMed][Google Scholar]
26. WongS.JamousA.O’DriscollJ.SekharR.WeldonM.YauC.Y.HiraniS.P.GrimbleG.ForbesA.2014A Lactobacillus casei Shirota probiotic drink reduces antibiotic-associateddiarrhoea in patients with spinal cord injuries: a randomised controlled trial. Br. J.Nutr. 111: 672–678[PubMed][Google Scholar]