BACKGROUND
inflammatory bowel disease  in dogs is a chronic gastrointestinal tract disorder of unknown cause  <cit> , although the mucosal immune system, genetic susceptibility, and the enteric microenvironment  are considered to be important in its pathogenesis .
treatment protocols for ibd combine diet modification, antibacterials and immunosuppressive therapy  <cit> , aiming at reducing intestinal inflammation and restoring gut normobiosis.
dietary modification is considered an adequate therapeutic starting point, since most ibd dogs respond positively to dietary management alone  <cit> .
dogs that do not respond or respond partially, require further treatment with antibiotics and/or immunosuppressive drugs like glucocorticoids, which can be associated with severe side effects .
ideally, any long-term treatment protocol for ibd should aim at maintaining patients in clinical remission for as long as possible by adequately combining such therapeutic modalities while intending to minimize associated side effects.
chondroitin sulfate , a natural glycosaminoglycan present in the extracellular matrix, has been suggested to reduce the incidence and severity of relapses in human ibd  <cit> .
cs inhibits nuclear factor kappa-light-chain-enhancer of activated b cells  activity  <cit> , which is also a suggested component of the anti-inflammatory activity of glucocorticoids and cyclosporine a  <cit> .
nf-κb is markedly increased in ibd  <cit> .
oral cs might therefore reduce intestinal inflammation and benefit dogs with ibd.
oxidative stress is also believed to play a key role in the pathogenesis of ibd  <cit> .
paraoxonase- <dig> , an antioxidant enzyme used as a biomarker of oxidative stress and inflammation in human ibd  <cit> , could also serve as a biomarker in dogs with ibd given the similarities between human and canine ibd.
in human ibd, lower pon <dig> serum concentrations are associated with increased inflammation and disease activity  <cit> .
decreasing oxidative stress, shown as an increased pon <dig>  could therefore be an effective endpoint for ibd treatment.
decreased serum tac levels have been reported in human patients with ulcerative colitis and crohn’s disease  <cit> .
lower bacterial diversity and altered microbial communities have been reported in canine ibd  <cit> .
orally administered prebiotics promote the growth of beneficial gut microbiota .
short chain fatty acids , especially butyrate, which is the preferential source of energy for colonocytes, help maintaining the intestinal mucosal barrier  <cit> .
a reduction in scfa producing bacteria, especially faecalibacterium spp.
and fusobacteria, has been reported in dogs with ibd  <cit> .
large-bowel fermentation of resistant starch increases butyrate production  <cit> .
consequently, oral administration of resistant starch could benefit ibd patients by increasing butyrate levels in their gut.
a dietary supplement containing cs and prebiotics ) was developed to target intestinal inflammation, oxidative stress and gut dysbiosis.
our research hypothesis was that long-term administration of this supplement is safe and helps to decrease intestinal inflammation and oxidative stress and restore normobiosis.
the purpose of this study was therefore to evaluate the effects of this supplement on clinical disease activity, intestinal histology, gut microbiota, and selected serum biomarkers in dogs with ibd over a time course of 180 days.
methods
dogs
this was a multicentric randomized double-blind placebo-controlled study using client-owned dogs.
all animal owners gave written informed consent.
the protocol was reviewed and approved by the animal experimentation committee of the complutense university of madrid .
both the owners and the investigators were blinded to treatment assignment until the end of the study.
inclusion criteria included dogs with a minimum age of 1 year, persistent  gastrointestinal signs, confirmation of ibd by histologic evaluation of biopsy samples and a baseline score of at least  <dig> on the canine ibd activity index   <cit> .
a diagnostic exclusion protocol was carried out in all patients in order to rule out other possible causes of chronic diarrhea, including urinalysis, abdominal ultrasound, fecal exam, cbc and serum biochemistry.
animals were excluded if they presented with significant extra-intestinal disease, intestinal neoplasia, or severe hypoproteinemia , if they had not been adequately dewormed within 60 days prior to trial entry, and if they had received antibiotics, prebiotics or probiotics 7 days prior to inclusion, or glucocorticoids 14 days prior to inclusion.
pregnant bitches were excluded.
treatment was initiated 15 days after endoscopy.
dogs with biopsy-confirmed ibd were then randomized in a 1: <dig> allocation ratio by means of a computer-generated schedule into one of the  <dig> treatment groups: placebo and supplement.
all dogs were switched to a hydrolyzed diet  which was maintained during the course of the study.
dogs in the placebo group  received a placebo powder  orally once daily, and dogs in the supplement <dig> group  received an oral daily dose per kg of bodyweight of 215 mg α-glucan butyrogenic resistant starch, 10 mg cs, and 26 mg β-glucans and mos for 180 days.
a rescue protocol was available in the case of clinical relapse; specifically, if the veterinarian responsible for the case decided that a considerable worsening had occurred since last visit, patients were administered a combination of metronidazole  for 21 days, ranitidine  and metoclopramide  for 60 days, and tapering doses of oral prednisone   <cit> .
relapsed cases that received the rescue protocol were excluded from the final data analysis.
evaluations
cibdai scores and serum biomarkers were monitored at the initial visit  and after  <dig>   <dig>   <dig>   <dig> and 180 days of treatment.
fasting blood samples were collected to measure serum biomarkers of inflammation ) and oxidative stress ).
in addition, serum albumin and cholesterol were measured.
the decrease in these two analytes in canine ibd has been associated with a worse histological score, because they are lost due to damaged intestinal villi and lacteals  <cit> .
albumin concentrations are also related to the severity of ibd in humans  <cit> , and in dogs  <cit> .
in human ibd patients, cholesterol levels show a negative correlation with disease activity  <cit> .
crp concentrations were measured using an immunoturbidimetric assay   <cit> .
pon <dig> activity was determined using p-nitrophenyl acetate as substrate  <cit> .
serum tac was measured by the method proposed by erel et al.
<cit>  with a minimum detection limit of  <dig>  mmol/l.
total cholesterol and albumin analyses were performed using commercially available kits.
all assays were performed in an automated clinical chemistry analyzer .
to assess changes in gut microbiota, fecal samples were analyzed by pcr-restriction fragment length polymorphism .
samples were collected at the initial visit and 120 days after treatment was initiated.
all analyses were performed by a single investigator .
immediately after collection, 1 g of fecal sample was placed in 3 ml of 98 % grade ethanol and cool transported to cresa lab ) where it was kept at 4 °c until dna extraction, as described elsewhere  <cit> .
a sample of 400 mg from the ethanol-preserved feces was washed twice with sterile buffered peptone water.
dna was extracted using the qiaamp dna stool minikit  following the manufacturer’s instructions with minor modifications including a lysozyme incubation step and adding ribonuclease-a and bsa  to the eluted dna  <cit> .
the 16s rdna was amplified as previously described  <cit> .
aliquots of the amplified dna fragments were digested in separate tubes with  <dig> restriction endonucleases .
the endonuclease fragments were resolved in 2 % agarose gels at 150 v for 60 min.
dna bands were visualized using an imaging system  and the genesnap software .
dendrograms showing the percentage of similarity among pcr-rflp band patterns were generated on the basis of the manhattan distance  <cit> .
in order to evaluate histologic changes, upper and lower gastrointestinal endoscopies were performed at the initial visit and after 180 days of treatment.
multiple biopsy samples from stomach ), small intestine  and large intestine  were obtained using different flexible video endoscopes depending on the size of the patient  and sent to citopath pathology service   for microscopic review.
samples were fixed in formalin, routinely processed, and embedded in paraffin.
serial sections from paraffin tissue blocks were cut for routine hematoxylin and eosin staining.
microscopic examination  of all tissues was performed by a single pathologist  who was blinded to treatments, and objectively graded endoscopic specimens following the wsava  histopathologic guidelines for the diagnosis of idiopathic inflammatory bowel diseases in dogs and cats  <cit> .
histologic scores for stomach, duodenum and colon were also evaluated and compared between groups.
concomitant treatments were recorded for all animals during the study, and relapse rates were compared between groups.
statistical analysis
statistical analysis was performed by a biostatistician  with the use of statistical software.
<dig> a one-way analysis of variance  of repeated measures and an uncorrected fisher’s least significant difference  test were used to compare the values obtained at different times during treatment with the pretreatment values.
unpaired t-test was performed to compare values between groups at each visit.
fisher’s exact test was performed to analyze differences in relapse rates between groups.
analytes that did not follow a parametric distribution were log-transformed before applying the test.
RESULTS
during the study period,  <dig> dogs were assessed for eligibility  and  <dig> of them were randomized into the clinical trial .
of the  <dig> dogs enrolled,  <dig> were randomized to group a  and  <dig> to group b .
breeds included yorkshire terrier , crossbreed , poodle , french bulldog , samoyed , german shepherd , and  <dig> dog each of the following breeds: basset hound, lhasa apso, great dane, siberian husky, shar-pei, chihuahua, beagle, and miniature schnauzer;  <dig> of the dogs were males and  <dig> were females, ranging in age from  <dig> to 15 years .
a total of four dogs  did not complete the study.
in group a, one dog discontinued the intervention due to owner decision; in group b,  <dig> dogs were excluded due to owner decision  and  <dig> dog was lost to follow-up.
overall,  <dig> dogs in group a  and  <dig> dogs in group b  completed the full clinical trial.fig.
<dig> flow diagram of ibd dogs.
group a, placebo; group b, supplement
canine ibd activity index
figure  <dig> shows changes in median cibdai scores.
at the time of diagnosis, there were no significant differences  between groups : placebo =  <dig> , supplement =  <dig> ).
after treatment, a significant reduction in median cibdai scores was observed in both groups .
compared to baseline median cibdai scores, the supplement group showed a 2-fold decrease  and a 4-fold decrease  after  <dig> and 60 days of treatment, respectively.
in the placebo group, decreases in median cibdai scores after  <dig> and 60 days of treatment were  <dig> -fold  and  <dig> -fold , respectively.
no significant differences between groups were found at any time point.fig.
<dig> one-way anova cibdai scores in group a  and b  15 days before treatment start , and at  <dig> ,  <dig> ,  <dig> ,  <dig>  and  <dig>  days of treatment.
asterisks indicate statistical significance compared to pretreatment values
serum biomarkers
initially, there were no statistically significant differences  between groups for any of the serum biomarkers analyzed.
initial median  values were within the reference ranges of our laboratory, with the exception of a decreased pon <dig> in all dogs ; supplement =  <dig>  ; reference values = 3– <dig>  iu/ml), increased crp in the placebo group ; reference values <12 μg/ml), and decreased tac in the supplement group ; reference values > <dig>  mm/l).
compared with pretreatment values, the supplement group showed significantly increased cholesterol and pon <dig> after  <dig> and 60 days of treatment  and a significantly decreased tac was observed in the placebo group from 90 days of treatment .
when groups were compared, the supplement group showed significantly higher cholesterol after  <dig> ,  <dig>  and  <dig>  days of treatment, and significantly higher  pon <dig> after  <dig>   <dig>   <dig> and 180 days of treatment compared to placebo.
during the study, in each group one dog showed cholesterol values above the reference range  and one below it.
pon <dig> remained below the reference range  in all dogs in the placebo group.
the placebo group showed significantly lower tac levels after 120 days of treatment  .
all dogs in the study showed tac values below the reference range  at some time point, but only with the supplement all dogs finished the study with levels within it.
no significant changes were seen for the other serum biomarkers.fig.
<dig> concentrations of serum cholesterol , pon <dig>  and tac  in groups a  and b  at the initial visit , and at  <dig> ,  <dig> ,  <dig> ,  <dig>  and  <dig>  days of treatment.
asterisks indicate statistical significance compared to pretreatment values within each group .
differences between groups are also represented
histologic examinations
variations in histologic scores are shown in fig.
<dig>  at the time of diagnosis, there were no significant differences between groups in overall wsava histologic scores  group a =  <dig> ; group b =  <dig> ; p =  <dig> ).
a second biopsy was not possible in  <dig> of the  <dig> patients that finished the study without needing the rescue protocol  due to reluctance of some owners to put their pet through another anesthetic procedure.
after treatment, the supplement group showed a significant  <dig> -fold decrease  in median overall histologic score, whereas a non-significant  <dig> -fold decrease  was observed in the placebo group.
with the supplement, a significant decrease was seen in duodenum  and colon .
no statistically significant differences were found with the placebo for any tissue.
when groups were compared, there were no significant differences at any time point.fig.
<dig> wsava histologic scores  in groups a  and b  at initial visit  and after 180 days of treatment  treatment.
asterisks indicate statistical significance compared to pretreatment  values
gut microbiota
the dendrogram formed with the band patterns generated by pcr-rflp of the fecal samples is shown in fig.
<dig>  no bands were generated in the pcr-rflp electrophoresis gel from samples belonging to two animals from different groups, probably due to an insufficient amount of dna.
consequently, these two study subjects were excluded from the dendrogram.
all but two samples were grouped in two main clusters at similarity levels of  <dig> and 72 %, respectively.
before and after treatment, samples from both the placebo and the supplement groups were not grouped together, but scattered throughout the dendrogram instead.
thus, no differences were observed between the placebo and the supplement groups or between initial and final samples of either group.fig.
<dig> ecological changes in microbial populations measured by pcr-rflp from fecal samples from groups a  and b  at initial visit  and at the end of the treatment .
the dendrogram illustrates the percentage of similarity among dna band patterns
clinical relapses and side effects
during the study, clinical relapses were observed in 3/ <dig> dogs in the placebo group  and in 1/ <dig> dogs in the supplement group .
no statistically significant differences were found in the proportion of animals that relapsed in each group .
in the placebo group, two dogs relapsed at  <dig> and 90 days of treatment and received the rescue protocol.
the third dog in the placebo group relapsed after 30 days of treatment, and received oral prednisone 1 mg/kg sid for 10 days.
in the supplement group, one case relapsed after 90 days of treatment and started the rescue treatment.
overall, both treatments were well tolerated and no side effects were reported in any of the groups.
discussion
the etiology of canine ibd is not fully understood, but there are some factors that are thought to be implicated in causing intestinal inflammation including an abnormal immune response, genetic susceptibility, nutrition and the intestinal microbiota .
herein, we report preliminary data on the efficacy and safety of a dietary supplement containing cs and prebiotics in dogs with ibd, which might suggest its usefulness to further extend clinical remission periods and avoid excessive usage of glucocorticoids and antibiotics, hence minimizing their unwanted side effects.
a significant decrease in cibdai was observed in both groups at the end of the study.
this clinical improvement regardless of the treatments received may be explained by a positive response to the hydrolyzed diet alone  <cit> .
to the authors’ knowledge, this is the first study to report measurements of pon <dig> in dogs with biopsy-confirmed ibd.
initially, serum values below our laboratory reference range for pon <dig> were found in all study dogs, which is in line with what has been observed in human patients with ibd  <cit> .
after treatment, an increased pon <dig> was observed only in the supplement group.
this could reflect a protective effect of the supplement against higher oxidative stress occurring in ibd.
in addition, in our study, serum cholesterol increased only in the supplement group.
this could indicate an improvement in the ibd since a decrease in serum cholesterol has been related to a worse histological score in dogs with ibd, probably because damaged intestinal villi and lacteals produce cholesterol loss  <cit> .
in human ibd patients, cholesterol levels show a negative correlation with disease activity and inflammatory indices  <cit> .
no significant changes in crp were observed after treatment in this study.
although increased concentrations of crp have been correlated with clinical disease activity in human patients with ibd and this marker is considered adequate for evaluating response to treatment in canine ibd  <cit> , other studies found that crp was not a good indicator of disease severity in dogs  <cit> .
we found significantly decreased tac values after treatment only with the placebo, which could indicate a higher susceptibility to oxidative tissue damage compared to the supplement group.
the differences found in our study between pon <dig> and tac might be explained by the fact that tac measures all the antioxidants of a sample, since it represents the measurement of the amount  of a free radical scavenged by a test solution  <cit> , and includes many antioxidants in addition to pon <dig>  therefore, depending on the behaviour of the different individual antioxidants, differences might appear between values of pon <dig>  and tac .
in addition, pon <dig> is also affected by inflammation in dogs  <cit> , which could also contribute to its different behaviour.
overall, serum results from this study could suggest a reduction of oxidative stress in the supplement group.
compared to baseline histologic scores, a significant improvement was seen only in the supplement group.
furthermore, these significant improvements were limited to the small and large intestines , which corresponds with the target tissues of the supplement.
nevertheless, the difference between groups in median wsava histologic score after treatment did not reach the level of statistical significance.
in our study, a clear change in microbial population was not seen in both groups despite the administration of prebiotics.
this could be attributable to the heterogeneity of our study subjects, which belonged to different breeds, age groups, and geographical regions with different environmental conditions.
it could also be related to the small sample size of the study.
moreover, this concurs with the findings of another recent study describing clinical improvements in dogs with ibd after medical therapy which were not accompanied by significant changes in their fecal microbiome  <cit> .
clinical relapses were seen earlier and more frequently in dogs belonging to the placebo group, although differences between groups were not statistically significant probably due to the study being underpowered.
although the results of the present study provide useful information about the effects of cs and prebiotics in dogs with ibd, there are some limitations that should be mentioned.
first, the sample size was small and this might have affected the level of statistical significance of certain parameters.
due to this concern, although better serological results were obtained with the supplement indicating and improved intestinal absorption capacity and decreased inflammation and oxidative stress, the observed improvements in histopathology and cibdai could not be statistically supported.
it should also be mentioned that male dogs and yorkshire terriers were overrepresented in this study, and that most cases came from one veterinary center .
lastly, pcr-rflp was selected as the technique for evaluating changes in gut microbiota.
ideally, other methods could have been used, but the authors chose pcr-rflp based on prior publications reporting its usefulness  <cit> .
CONCLUSIONS
our results suggest a beneficial effect of long-term oral administration of a dietary supplement containing cs and prebiotics combined with a hydrolyzed diet in dogs with ibd by increasing serum pon <dig>  tac and cholesterol in dogs with ibd.
the administration of the supplement over 180 days was safe.
a supplemental effect to dietary treatment was not statistically demonstrated for cibdai, histology and relapse rates, likely because the study was underpowered.
further studies with larger randomized clinical trials are warranted to confirm these results.
abbreviations
ibdinflammatory bowel disease
cschondroitin sulfate
nf-κbnuclear factor kappa-light-chain-enhancer of activated b cells
pon1paraoxonase-1
scfashort chain fatty acids
mosmannanoligosaccharides
ucmcomplutense university of madrid
cibdaicanine ibd activity index
crpc-reactive protein
tactotal antioxidant capacity
pcr-rflppcr-restriction fragment length polymorphism
wsavaworld small animal veterinary association
anovaanalysis of variance
lsdleast significant difference
<dig> entero-chronic®, bioiberica sa, barcelona, spain.
<dig> graphpad prism  <dig>  graphpad software, inc, san diego, usa.
competing interests
ss, dmp and av are employed by bioiberica sa, spain.
bioiberica sa provided the compounds used in the study and funding for this clinical trial.
ka and jc do scientific consultancy for bioiberica sa.
authors’ contributions
ss, dmp, frf and av participated in the design of the study.
ss supervised the study, participated in data analysis and interpretation of results, prepared the draft of the manuscript and submitted the final manuscript.
sms performed the statistical analysis.
mcc performed the pcr-rflp analyses of fecal samples.
sms, amp and jc carried out the serum samples analysis.
arb conducted the histopathological evaluations.
ka and jc collaborated on data analysis and interpretation of results, and critically revised the manuscript.
all authors read and approved the final manuscript.
the authors would like to thank verena blume and míriam sellés for their time and effort, as well as the veterinary centers that participated in the study: cpv raspeig, alicante; cv mediterráneo, madrid; hv tomás bustamante, cantabria; hv los madrazo, madrid; cv estoril, madrid; hv guadiamar, sevilla; hcv complutense, madrid; and hv canis i felis, barcelona.
technical support from teresa ayats  is also acknowledged.

 inflammatory bowel disease in dogs is a chronic gastrointestinal tract disorder of unknown cause, although the mucosal immune system, genetic susceptibility, and the enteric microenvironment are considered to be important in its pathogenesis. treatment protocols for ibd combine diet modification, antibacterials and immunosuppressive therapy, aiming at reducing intestinal inflammation and restoring gut normobiosis. dietary modification is considered an adequate therapeutic starting point, since most ibd dogs respond positively to dietary management alone. dogs that do not respond or respond partially, require further treatment with antibiotics and/or immunosuppressive drugs like glucocorticoids, which can be associated with severe side effects. ideally, any long-term treatment protocol for ibd should aim at maintaining patients in clinical remission for as long as possible by adequately combining such therapeutic modalities while intending to minimize associated side effects. chondroitin sulfate, a natural glycosaminoglycan present in the extracellular matrix, has been suggested to reduce the incidence and severity of relapses in human ibd. cs inhibits nuclear factor kappa-light-chain-enhancer of activated b cells activity, which is also a suggested component of the anti-inflammatory activity of glucocorticoids and cyclosporine a. nf-κb is markedly increased in ibd. oral cs might therefore reduce intestinal inflammation and benefit dogs with ibd. oxidative stress is also believed to play a key role in the pathogenesis of ibd. paraoxonase-, an antioxidant enzyme used as a biomarker of oxidative stress and inflammation in human ibd, could also serve as a biomarker in dogs with ibd given the similarities between human and canine ibd. in human ibd, lower pon serum concentrations are associated with increased inflammation and disease activity. decreasing oxidative stress, shown as an increased pon could therefore be an effective endpoint for ibd treatment. decreased serum tac levels have been reported in human patients with ulcerative colitis and crohn’s disease. lower bacterial diversity and altered microbial communities have been reported in canine ibd. orally administered prebiotics promote the growth of beneficial gut microbiota. short chain fatty acids, especially butyrate, which is the preferential source of energy for colonocytes, help maintaining the intestinal mucosal barrier. a reduction in scfa producing bacteria, especially faecalibacterium spp. and fusobacteria, has been reported in dogs with ibd. large-bowel fermentation of resistant starch increases butyrate production. consequently, oral administration of resistant starch could benefit ibd patients by increasing butyrate levels in their gut. a dietary supplement containing cs and prebiotics ) was developed to target intestinal inflammation, oxidative stress and gut dysbiosis. our research hypothesis was that long-term administration of this supplement is safe and helps to decrease intestinal inflammation and oxidative stress and restore normobiosis. the purpose of this study was therefore to evaluate the effects of this supplement on clinical disease activity, intestinal histology, gut microbiota, and selected serum biomarkers in dogs with ibd over a time course of 180 days. methods dogs this was a multicentric randomized double-blind placebo-controlled study using client-owned dogs. all animal owners gave written informed consent. the protocol was reviewed and approved by the animal experimentation committee of the complutense university of madrid. both the owners and the investigators were blinded to treatment assignment until the end of the study. inclusion criteria included dogs with a minimum age of 1 year, persistent gastrointestinal signs, confirmation of ibd by histologic evaluation of biopsy samples and a baseline score of at least on the canine ibd activity index. a diagnostic exclusion protocol was carried out in all patients in order to rule out other possible causes of chronic diarrhea, including urinalysis, abdominal ultrasound, fecal exam, cbc and serum biochemistry. animals were excluded if they presented with significant extra-intestinal disease, intestinal neoplasia, or severe hypoproteinemia, if they had not been adequately dewormed within 60 days prior to trial entry, and if they had received antibiotics, prebiotics or probiotics 7 days prior to inclusion, or glucocorticoids 14 days prior to inclusion. pregnant bitches were excluded. treatment was initiated 15 days after endoscopy. dogs with biopsy-confirmed ibd were then randomized in a 1: allocation ratio by means of a computer-generated schedule into one of the treatment groups: placebo and supplement. all dogs were switched to a hydrolyzed diet which was maintained during the course of the study. dogs in the placebo group received a placebo powder orally once daily, and dogs in the supplement group received an oral daily dose per kg of bodyweight of 215 mg α-glucan butyrogenic resistant starch, 10 mg cs, and 26 mg β-glucans and mos for 180 days. a rescue protocol was available in the case of clinical relapse; specifically, if the veterinarian responsible for the case decided that a considerable worsening had occurred since last visit, patients were administered a combination of metronidazole for 21 days, ranitidine and metoclopramide for 60 days, and tapering doses of oral prednisone. relapsed cases that received the rescue protocol were excluded from the final data analysis. evaluations cibdai scores and serum biomarkers were monitored at the initial visit and after and 180 days of treatment. fasting blood samples were collected to measure serum biomarkers of inflammation ) and oxidative stress ). in addition, serum albumin and cholesterol were measured. the decrease in these two analytes in canine ibd has been associated with a worse histological score, because they are lost due to damaged intestinal villi and lacteals. albumin concentrations are also related to the severity of ibd in humans, and in dogs. in human ibd patients, cholesterol levels show a negative correlation with disease activity. crp concentrations were measured using an immunoturbidimetric assay. pon activity was determined using p-nitrophenyl acetate as substrate. serum tac was measured by the method proposed by erel et al. with a minimum detection limit of  mmol/l. total cholesterol and albumin analyses were performed using commercially available kits. all assays were performed in an automated clinical chemistry analyzer. to assess changes in gut microbiota, fecal samples were analyzed by pcr-restriction fragment length polymorphism. samples were collected at the initial visit and 120 days after treatment was initiated. all analyses were performed by a single investigator. immediately after collection, 1 g of fecal sample was placed in 3 ml of 98 % grade ethanol and cool transported to cresa lab ) where it was kept at 4 °c until dna extraction, as described elsewhere. a sample of 400 mg from the ethanol-preserved feces was washed twice with sterile buffered peptone water. dna was extracted using the qiaamp dna stool minikit following the manufacturer’s instructions with minor modifications including a lysozyme incubation step and adding ribonuclease-a and bsa to the eluted dna. the 16s rdna was amplified as previously described. aliquots of the amplified dna fragments were digested in separate tubes with restriction endonucleases. the endonuclease fragments were resolved in 2 % agarose gels at 150 v for 60 min. dna bands were visualized using an imaging system and the genesnap software. dendrograms showing the percentage of similarity among pcr-rflp band patterns were generated on the basis of the manhattan distance. in order to evaluate histologic changes, upper and lower gastrointestinal endoscopies were performed at the initial visit and after 180 days of treatment. multiple biopsy samples from stomach ), small intestine and large intestine were obtained using different flexible video endoscopes depending on the size of the patient and sent to citopath pathology service for microscopic review. samples were fixed in formalin, routinely processed, and embedded in paraffin. serial sections from paraffin tissue blocks were cut for routine hematoxylin and eosin staining. microscopic examination of all tissues was performed by a single pathologist who was blinded to treatments, and objectively graded endoscopic specimens following the wsava histopathologic guidelines for the diagnosis of idiopathic inflammatory bowel diseases in dogs and cats. histologic scores for stomach, duodenum and colon were also evaluated and compared between groups. concomitant treatments were recorded for all animals during the study, and relapse rates were compared between groups. statistical analysis statistical analysis was performed by a biostatistician with the use of statistical software. a one-way analysis of variance of repeated measures and an uncorrected fisher’s least significant difference test were used to compare the values obtained at different times during treatment with the pretreatment values. unpaired t-test was performed to compare values between groups at each visit. fisher’s exact test was performed to analyze differences in relapse rates between groups. analytes that did not follow a parametric distribution were log-transformed before applying the test.  during the study period, dogs were assessed for eligibility and of them were randomized into the clinical trial. of the dogs enrolled, were randomized to group a and to group b. breeds included yorkshire terrier, crossbreed, poodle, french bulldog, samoyed, german shepherd, and dog each of the following breeds: basset hound, lhasa apso, great dane, siberian husky, shar-pei, chihuahua, beagle, and miniature schnauzer; of the dogs were males and were females, ranging in age from to 15 years. a total of four dogs did not complete the study. in group a, one dog discontinued the intervention due to owner decision; in group b, dogs were excluded due to owner decision and dog was lost to follow-up. overall, dogs in group a and dogs in group b completed the full clinical trial.fig. flow diagram of ibd dogs. group a, placebo; group b, supplement canine ibd activity index figure  shows changes in median cibdai scores. at the time of diagnosis, there were no significant differences between groups: placebo = , supplement =  ). after treatment, a significant reduction in median cibdai scores was observed in both groups. compared to baseline median cibdai scores, the supplement group showed a 2-fold decrease and a 4-fold decrease after and 60 days of treatment, respectively. in the placebo group, decreases in median cibdai scores after and 60 days of treatment were -fold and -fold, respectively. no significant differences between groups were found at any time point.fig. one-way anova cibdai scores in group a and b 15 days before treatment start, and at,,, and days of treatment. asterisks indicate statistical significance compared to pretreatment values serum biomarkers initially, there were no statistically significant differences between groups for any of the serum biomarkers analyzed. initial median values were within the reference ranges of our laboratory, with the exception of a decreased pon in all dogs; supplement = ; reference values = 3–  iu/ml), increased crp in the placebo group; reference values <12 μg/ml), and decreased tac in the supplement group; reference values >  mm/l). compared with pretreatment values, the supplement group showed significantly increased cholesterol and pon after and 60 days of treatment and a significantly decreased tac was observed in the placebo group from 90 days of treatment. when groups were compared, the supplement group showed significantly higher cholesterol after, and days of treatment, and significantly higher pon after and 180 days of treatment compared to placebo. during the study, in each group one dog showed cholesterol values above the reference range and one below it. pon remained below the reference range in all dogs in the placebo group. the placebo group showed significantly lower tac levels after 120 days of treatment. all dogs in the study showed tac values below the reference range at some time point, but only with the supplement all dogs finished the study with levels within it. no significant changes were seen for the other serum biomarkers.fig. concentrations of serum cholesterol, pon and tac in groups a and b at the initial visit, and at,,, and days of treatment. asterisks indicate statistical significance compared to pretreatment values within each group. differences between groups are also represented histologic examinations variations in histologic scores are shown in fig.  at the time of diagnosis, there were no significant differences between groups in overall wsava histologic scores group a = ; group b = ; p =  ). a second biopsy was not possible in of the patients that finished the study without needing the rescue protocol due to reluctance of some owners to put their pet through another anesthetic procedure. after treatment, the supplement group showed a significant -fold decrease in median overall histologic score, whereas a non-significant -fold decrease was observed in the placebo group. with the supplement, a significant decrease was seen in duodenum and colon. no statistically significant differences were found with the placebo for any tissue. when groups were compared, there were no significant differences at any time point.fig. wsava histologic scores in groups a and b at initial visit and after 180 days of treatment treatment. asterisks indicate statistical significance compared to pretreatment values gut microbiota the dendrogram formed with the band patterns generated by pcr-rflp of the fecal samples is shown in fig.  no bands were generated in the pcr-rflp electrophoresis gel from samples belonging to two animals from different groups, probably due to an insufficient amount of dna. consequently, these two study subjects were excluded from the dendrogram. all but two samples were grouped in two main clusters at similarity levels of and 72 %, respectively. before and after treatment, samples from both the placebo and the supplement groups were not grouped together, but scattered throughout the dendrogram instead. thus, no differences were observed between the placebo and the supplement groups or between initial and final samples of either group.fig. ecological changes in microbial populations measured by pcr-rflp from fecal samples from groups a and b at initial visit and at the end of the treatment. the dendrogram illustrates the percentage of similarity among dna band patterns clinical relapses and side effects during the study, clinical relapses were observed in 3/ dogs in the placebo group and in 1/ dogs in the supplement group. no statistically significant differences were found in the proportion of animals that relapsed in each group. in the placebo group, two dogs relapsed at and 90 days of treatment and received the rescue protocol. the third dog in the placebo group relapsed after 30 days of treatment, and received oral prednisone 1 mg/kg sid for 10 days. in the supplement group, one case relapsed after 90 days of treatment and started the rescue treatment. overall, both treatments were well tolerated and no side effects were reported in any of the groups. discussion the etiology of canine ibd is not fully understood, but there are some factors that are thought to be implicated in causing intestinal inflammation including an abnormal immune response, genetic susceptibility, nutrition and the intestinal microbiota. herein, we report preliminary data on the efficacy and safety of a dietary supplement containing cs and prebiotics in dogs with ibd, which might suggest its usefulness to further extend clinical remission periods and avoid excessive usage of glucocorticoids and antibiotics, hence minimizing their unwanted side effects. a significant decrease in cibdai was observed in both groups at the end of the study. this clinical improvement regardless of the treatments received may be explained by a positive response to the hydrolyzed diet alone. to the authors’ knowledge, this is the first study to report measurements of pon in dogs with biopsy-confirmed ibd. initially, serum values below our laboratory reference range for pon were found in all study dogs, which is in line with what has been observed in human patients with ibd. after treatment, an increased pon was observed only in the supplement group. this could reflect a protective effect of the supplement against higher oxidative stress occurring in ibd. in addition, in our study, serum cholesterol increased only in the supplement group. this could indicate an improvement in the ibd since a decrease in serum cholesterol has been related to a worse histological score in dogs with ibd, probably because damaged intestinal villi and lacteals produce cholesterol loss. in human ibd patients, cholesterol levels show a negative correlation with disease activity and inflammatory indices. no significant changes in crp were observed after treatment in this study. although increased concentrations of crp have been correlated with clinical disease activity in human patients with ibd and this marker is considered adequate for evaluating response to treatment in canine ibd, other studies found that crp was not a good indicator of disease severity in dogs. we found significantly decreased tac values after treatment only with the placebo, which could indicate a higher susceptibility to oxidative tissue damage compared to the supplement group. the differences found in our study between pon and tac might be explained by the fact that tac measures all the antioxidants of a sample, since it represents the measurement of the amount of a free radical scavenged by a test solution, and includes many antioxidants in addition to pon therefore, depending on the behaviour of the different individual antioxidants, differences might appear between values of pon and tac. in addition, pon is also affected by inflammation in dogs, which could also contribute to its different behaviour. overall, serum results from this study could suggest a reduction of oxidative stress in the supplement group. compared to baseline histologic scores, a significant improvement was seen only in the supplement group. furthermore, these significant improvements were limited to the small and large intestines, which corresponds with the target tissues of the supplement. nevertheless, the difference between groups in median wsava histologic score after treatment did not reach the level of statistical significance. in our study, a clear change in microbial population was not seen in both groups despite the administration of prebiotics. this could be attributable to the heterogeneity of our study subjects, which belonged to different breeds, age groups, and geographical regions with different environmental conditions. it could also be related to the small sample size of the study. moreover, this concurs with the findings of another recent study describing clinical improvements in dogs with ibd after medical therapy which were not accompanied by significant changes in their fecal microbiome. clinical relapses were seen earlier and more frequently in dogs belonging to the placebo group, although differences between groups were not statistically significant probably due to the study being underpowered. although the results of the present study provide useful information about the effects of cs and prebiotics in dogs with ibd, there are some limitations that should be mentioned. first, the sample size was small and this might have affected the level of statistical significance of certain parameters. due to this concern, although better serological results were obtained with the supplement indicating and improved intestinal absorption capacity and decreased inflammation and oxidative stress, the observed improvements in histopathology and cibdai could not be statistically supported. it should also be mentioned that male dogs and yorkshire terriers were overrepresented in this study, and that most cases came from one veterinary center. lastly, pcr-rflp was selected as the technique for evaluating changes in gut microbiota. ideally, other methods could have been used, but the authors chose pcr-rflp based on prior publications reporting its usefulness.  our results suggest a beneficial effect of long-term oral administration of a dietary supplement containing cs and prebiotics combined with a hydrolyzed diet in dogs with ibd by increasing serum pon tac and cholesterol in dogs with ibd. the administration of the supplement over 180 days was safe. a supplemental effect to dietary treatment was not statistically demonstrated for cibdai, histology and relapse rates, likely because the study was underpowered. further studies with larger randomized clinical trials are warranted to confirm these results. abbreviations ibdinflammatory bowel disease cschondroitin sulfate nf-κbnuclear factor kappa-light-chain-enhancer of activated b cells pon1paraoxonase-1 scfashort chain fatty acids mosmannanoligosaccharides ucmcomplutense university of madrid cibdaicanine ibd activity index crpc-reactive protein tactotal antioxidant capacity pcr-rflppcr-restriction fragment length polymorphism wsavaworld small animal veterinary association anovaanalysis of variance lsdleast significant difference entero-chronic®, bioiberica sa, barcelona, spain. graphpad prism graphpad software, inc, san diego, usa. competing interests ss, dmp and av are employed by bioiberica sa, spain. bioiberica sa provided the compounds used in the study and funding for this clinical trial. ka and jc do scientific consultancy for bioiberica sa. authors’ contributions ss, dmp, frf and av participated in the design of the study. ss supervised the study, participated in data analysis and interpretation of results, prepared the draft of the manuscript and submitted the final manuscript. sms performed the statistical analysis. mcc performed the pcr-rflp analyses of fecal samples. sms, amp and jc carried out the serum samples analysis. arb conducted the histopathological evaluations. ka and jc collaborated on data analysis and interpretation of results, and critically revised the manuscript. all authors read and approved the final manuscript. the authors would like to thank verena blume and míriam sellés for their time and effort, as well as the veterinary centers that participated in the study: cpv raspeig, alicante; cv mediterráneo, madrid; hv tomás bustamante, cantabria; hv los madrazo, madrid; cv estoril, madrid; hv guadiamar, sevilla; hcv complutense, madrid; and hv canis i felis, barcelona. technical support from teresa ayats is also acknowledged.
this study was likely underpowered, therefore larger studies are warranted in order to demonstrate a supplemental effect to dietary treatment of this supplement on intestinal histology and cibdai. when groups were compared, the supplement group showed significantly higher serum cholesterol and paraoxonase- levels after 60 days of treatment, and the placebo group showed significantly reduced serum total antioxidant capacity levels after 120 days. a new oral supplement for long-term management of canine ibd containing chondroitin sulfate and prebiotics was developed to target intestinal inflammation and oxidative stress, and restore normobiosis, without exhibiting any side effects. this double-blinded, randomized, placebo-controlled trial in dogs with ibd aims to evaluate the effects of 180 days administration of this supplement together with a hydrolyzed diet on clinical signs, intestinal histology, gut microbiota, and serum biomarkers of inflammation and oxidative stress. final data analysis showed a significant decrease in canine ibd activity index score in both groups after treatment. no significant differences were found between groups at any time point for cibdai, wsava histologic score and fecal microbiota evaluated by pcr-restriction fragment length polymorphism. since some of the current therapies are associated with severe side effects, novel therapeutic modalities are needed. twenty-seven client-owned biopsy-confirmed ibd dogs were included in the study, switched to the same hydrolyzed diet and classified into one of two groups: supplement and placebo. initially, there were no significant differences between groups for any of the studied parameters. the combined administration of the supplement with hydrolyzed diet over 180 days was safe and induced improvements in selected serum biomarkers, possibly suggesting a reduction in disease activity. canine inflammatory bowel disease is a chronic enteropathy of unknown etiology, although microbiome dysbiosis, genetic susceptibility, and dietary and/or environmental factors are hypothesized to be involved in its pathogenesis. chronic enteropathydogglycosaminoglycanresistant starchβ-glucansoxidative stresscibdaibioiberica saissue-copyright-statement© the author 2016
 canine inflammatory bowel disease is a chronic enteropathy of unknown etiology, although microbiome dysbiosis, genetic susceptibility, and dietary and/or environmental factors are hypothesized to be involved in its pathogenesis. since some of the current therapies are associated with severe side effects, novel therapeutic modalities are needed. a new oral supplement for long-term management of canine ibd containing chondroitin sulfate and prebiotics was developed to target intestinal inflammation and oxidative stress, and restore normobiosis, without exhibiting any side effects. this double-blinded, randomized, placebo-controlled trial in dogs with ibd aims to evaluate the effects of 180 days administration of this supplement together with a hydrolyzed diet on clinical signs, intestinal histology, gut microbiota, and serum biomarkers of inflammation and oxidative stress.  twenty-seven client-owned biopsy-confirmed ibd dogs were included in the study, switched to the same hydrolyzed diet and classified into one of two groups: supplement and placebo. initially, there were no significant differences between groups for any of the studied parameters. final data analysis showed a significant decrease in canine ibd activity index score in both groups after treatment. after treatment, a significant decrease in histologic score was seen only in the supplement group. when groups were compared, the supplement group showed significantly higher serum cholesterol and paraoxonase- levels after 60 days of treatment, and the placebo group showed significantly reduced serum total antioxidant capacity levels after 120 days. no significant differences were found between groups at any time point for cibdai, wsava histologic score and fecal microbiota evaluated by pcr-restriction fragment length polymorphism. no side effects were reported in any group.  the combined administration of the supplement with hydrolyzed diet over 180 days was safe and induced improvements in selected serum biomarkers, possibly suggesting a reduction in disease activity. this study was likely underpowered, therefore larger studies are warranted in order to demonstrate a supplemental effect to dietary treatment of this supplement on intestinal histology and cibdai. keywords chronic enteropathydogglycosaminoglycanresistant starchβ-glucansoxidative stresscibdaibioiberica saissue-copyright-statement© the author 2016 