The induction of GIT inflammation in mice via administration of dextran sulfate sodium (DSS) in the drinking water for seven days was associated with increased numbers of aerobic bacteria, specifically *Entercococcus faecalis*[210]. Bacteroidetes were eliminated from the community and the total number of bacteria was also decreased. A dramatic increase in the numbers of a nonpathogenic *E. coli* following DSS treatment was also observed. The authors subsequently colonized IL-10-/- mice, which spontaneously develop colitis [213], with this non-pathogenic *E. coli*. Upon the development of GIT inflammation, the *E. coli* was able to proliferate to the same level observed in DSS treated mice, but displacing the Firmicutes phylum instead of the Bacteroidetes. These results indicate that regardless of the source, GIT inflammation creates a niche that favors the colonization and expansion of *Enterobacteriaceae.* Collectively, these data suggest that the induction of inflammation following the colonization of the GIT by a bacterial provocateur along with perturbations of the microbiota together have a deleterious impact on the microbiota and mucosal homeostasis.

Mutations in innate immune receptors in German shepherd dogs (TLR5, NOD2) have been linked to IBD susceptibility which in the presence of an inappropriate enteric microbiota may lead to upregulated pro-inflammatory cytokine production (e.g., IL-17, IL-22, TNF-α) and reduced bacterial clearance, thereby promoting chronic intestinal inflammation [218, 219]. Commensal bacterial antigens are likely to be important in disease pathogenesis because it has been observed that boxer dogs with granulomatous colitis (GC) show clinical remission following the eradication of mucosally associated AIEC that share a novel adherent and invasive pathotype which bears phylogenetic similarity with AIEC strains recovered from patients with ileal Crohn's disease [193, 220, 221]. Moreover, genome-wide analysis in affected boxer dogs has identified disease-associated single nucleotide polymorphisms (SNPs) in a gene (*NCF2*) involved with killing intracellular bacteria [222]. Still others have shown that

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cells are significantly decreased in the intestines of dogs with IBD suggesting that

chronic mucosal inflammation may involve an imbalance in the intestinal dendritic cell population leading to aberrant immune activation [223]. Molecular analysis of the intestinal microbiome in different breeds of dogs with IBD have consistently shown that diseased tissues are enriched with members of the families *Enterobacteriaceae* and *Clostridiaceae*[224, 225]. These bacteria are believed to contribute to the pathogenesis of GIT inflammatory disease in dogs as in humans [187, 226]. A recent trial using high throughput 16S rDNA sequencing methods (ie, 454 pyrosequencing) on intestinal biopsies of IBD dogs revealed a dysbiosis in the mucosallyadherent microbiota with an increase in sequences belonging to Proteobacteria and a decrease in Bacteroidetes, Fusobacteria, and the Clostridiales [227]. Taken together, these studies suggest that chronic intestinal inflammation of canine IBD may be due to overly aggressive adaptive immune responses to enteric bacteria (or fungi) [225] in hosts with genetic defects that fail to properly regulate microbial killing, mucosal barrier function, or immune responses. As in human IBD, environmental factors (diet, microbiota imbalances) likely govern the onset

of inflammation or reactivation and modulate genetic susceptibility to disease.

The clinical manifestations of IBD are diverse and are influenced by the organ(s) involved, presence of active versus inactive disease, and physiologic complications seen with enteric plasma protein loss and/or micronutrient (cobalamin) deficiency [215, 216, 228, 229]. Canine IBD is a disease that predominantly affects middle-aged animals. Vomiting and diarrhea are most commonly observed and are often accompanied by decreased appetite and weight loss. Gastric and duodenal inflammation is associated with vomiting and small bowel diarrhea while colonic involvement causes large bowel diarrhea with blood, mucus, and straining. The clinical course of IBD is generally cyclical and is characterized by spontaneous exacerbations and remissions. Importantly, the clinical signs of IBD are not disease specific and share numerous over-lapping features with other canine disorders. A diagnosis of IBD is one of exclusion and requires careful elimination of IBD mimics [230]. The possible causes for chronic intestinal inflammation may be excluded through the integration of history, physical findings, clinicopathological testing, diagnostic imaging, and histopathology of intestinal biopsies. A baseline CBC, biochemistry profile, urinalysis, and diagnostic imaging are useful in eliminat‐ ing the most common systemic and metabolic disorders (e.g., renal disease, hepatopathy,

*2.5.1. Clinical and diagnostic features*

CD11c+

## **2.5. Canine IBD**

In addition to affecting humans, IBD can also occur in dogs as well [214]. As in human IBD, the interactions between genetics, the mucosal immune system, inflammation, and environ‐ mental factors (ie, diet and imbalances in the intestinal microbiome) all contribute to the pathogenesis of canine IBD (Figure 6) [215-217].

**Figure 6.** The etiology for canine IBD involves complex interactions between host genetics, mucosal immunity, and the enteric microbiota. Therapeutic intervention with diet, antibiotics and immunosuppressive drugs is aimed at re‐ ducing inflammation and dysbiosis.

Mutations in innate immune receptors in German shepherd dogs (TLR5, NOD2) have been linked to IBD susceptibility which in the presence of an inappropriate enteric microbiota may lead to upregulated pro-inflammatory cytokine production (e.g., IL-17, IL-22, TNF-α) and reduced bacterial clearance, thereby promoting chronic intestinal inflammation [218, 219]. Commensal bacterial antigens are likely to be important in disease pathogenesis because it has been observed that boxer dogs with granulomatous colitis (GC) show clinical remission following the eradication of mucosally associated AIEC that share a novel adherent and invasive pathotype which bears phylogenetic similarity with AIEC strains recovered from patients with ileal Crohn's disease [193, 220, 221]. Moreover, genome-wide analysis in affected boxer dogs has identified disease-associated single nucleotide polymorphisms (SNPs) in a gene (*NCF2*) involved with killing intracellular bacteria [222]. Still others have shown that CD11c+ cells are significantly decreased in the intestines of dogs with IBD suggesting that chronic mucosal inflammation may involve an imbalance in the intestinal dendritic cell population leading to aberrant immune activation [223]. Molecular analysis of the intestinal microbiome in different breeds of dogs with IBD have consistently shown that diseased tissues are enriched with members of the families *Enterobacteriaceae* and *Clostridiaceae*[224, 225]. These bacteria are believed to contribute to the pathogenesis of GIT inflammatory disease in dogs as in humans [187, 226]. A recent trial using high throughput 16S rDNA sequencing methods (ie, 454 pyrosequencing) on intestinal biopsies of IBD dogs revealed a dysbiosis in the mucosallyadherent microbiota with an increase in sequences belonging to Proteobacteria and a decrease in Bacteroidetes, Fusobacteria, and the Clostridiales [227]. Taken together, these studies suggest that chronic intestinal inflammation of canine IBD may be due to overly aggressive adaptive immune responses to enteric bacteria (or fungi) [225] in hosts with genetic defects that fail to properly regulate microbial killing, mucosal barrier function, or immune responses. As in human IBD, environmental factors (diet, microbiota imbalances) likely govern the onset of inflammation or reactivation and modulate genetic susceptibility to disease.

#### *2.5.1. Clinical and diagnostic features*

provocateur along with perturbations of the microbiota together have a deleterious impact on

In addition to affecting humans, IBD can also occur in dogs as well [214]. As in human IBD, the interactions between genetics, the mucosal immune system, inflammation, and environ‐ mental factors (ie, diet and imbalances in the intestinal microbiome) all contribute to the

**Figure 6.** The etiology for canine IBD involves complex interactions between host genetics, mucosal immunity, and the enteric microbiota. Therapeutic intervention with diet, antibiotics and immunosuppressive drugs is aimed at re‐

the microbiota and mucosal homeostasis.

pathogenesis of canine IBD (Figure 6) [215-217].

**2.5. Canine IBD**

94 Inflammatory Bowel Disease

ducing inflammation and dysbiosis.

The clinical manifestations of IBD are diverse and are influenced by the organ(s) involved, presence of active versus inactive disease, and physiologic complications seen with enteric plasma protein loss and/or micronutrient (cobalamin) deficiency [215, 216, 228, 229]. Canine IBD is a disease that predominantly affects middle-aged animals. Vomiting and diarrhea are most commonly observed and are often accompanied by decreased appetite and weight loss. Gastric and duodenal inflammation is associated with vomiting and small bowel diarrhea while colonic involvement causes large bowel diarrhea with blood, mucus, and straining. The clinical course of IBD is generally cyclical and is characterized by spontaneous exacerbations and remissions. Importantly, the clinical signs of IBD are not disease specific and share numerous over-lapping features with other canine disorders. A diagnosis of IBD is one of exclusion and requires careful elimination of IBD mimics [230]. The possible causes for chronic intestinal inflammation may be excluded through the integration of history, physical findings, clinicopathological testing, diagnostic imaging, and histopathology of intestinal biopsies. A baseline CBC, biochemistry profile, urinalysis, and diagnostic imaging are useful in eliminat‐ ing the most common systemic and metabolic disorders (e.g., renal disease, hepatopathy, hypoadrenocorticism) causing chronic GI signs in dogs. The measure of clinical disease activity by means of quantifiable indices is well established in human IBD [231-233].

tion and maintenance of remission of clinical signs have not been determined for most protocols [216, 230]. In general, the administration of corticosteroids (i.e., prednisone, prednisolone or budesonide), antimicrobials (i.e., metronidazole or tylosin), and immuno‐ suppressive drugs (i.e., cyclosporine, chlorambucil, azathioprine) used alone or in some combination are effective in inducing clinical remission in most animals. Some dogs will

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The rationale for nutritional therapy of IBD is that restricting exposure to antigens (i.e., dietary proteins) known to evoke sensitivity will reduce exaggerated host responses and attenuate intestinal inflammation. Other indications for specialized nutrition include the presence of decreased appetite, impaired nutrient absorption, or enteric plasma protein loss seen with moderate-to-severe mucosal inflammation. While evidence-based observations indicate that most dogs respond favorably to dietary intervention, the superiority of one novel protein source versus another or the advantage in feeding an intact protein elimination diet versus a hydrolyzed protein elimination diet has not been shown to date. Modifying the dietary n3:n6 fatty-acid ratio may also modulate inflammatory responses by reducing production of proinflammatory metabolites [237]. There is relatively sparse clinical data investigating prebiotic

Canine IBD represents a common and frustrating GI disorder in veterinary medicine. More research is needed to unravel the mechanisms responsible for disease development and to translate these findings directly to human IBD. The primary features of IBD in humans and

> **Feature Human IBD Canine IBD** Genetic basis Yes Likely

> > pANCA, CRP, calprotectin)

Involves the microbiota Yes Yes Hematochezia Yes Yes Diarrhea Yes Yes Definitive Diagnosis GI biopsy GI biopsy

Responsive to anti-inflammatory drugs Yes Yes

Responsive to antibiotics Yes Yes Spontaneous GI flares Yes Yes

Disease activity assessment Clinical indices, biomarkers (ASCA,

**Table 1.** Comparative features of IBD in humans and dogs.

Etiology Unknown but multifactorial Unknown but multifactorial

Clinical indices, biomarkers (pANCA, CRP, calprotectin ?)

or probiotic therapy for canine IBD (see subsequent section on probiotics).

require intermittent or life-long drug therapy.

*2.5.3. Future directions in canine IBD*

animals are remarkably similar (Table 1).

A canine IBD activity index (CIBDAI) used for assessment of inflammatory activity in dogs has been recently designed [234]. Similar to other indices, the magnitude of the numerical score is proportional to the degree of inflammatory activity. This index serves as the principal measure of response to a therapeutic regimen and may be used to tailor medical therapy for an individual patient's needs [235]. Intestinal biopsies are required to confirm histopatholog‐ ical inflammation and to determine the extent of mucosal disease. Diagnostic endoscopy is preferred since this technique allows for direct assessment of mucosal abnormalities and the acquisition of targeted biopsy specimens. The microscopic findings in canine IBD consist of minimal to pronounced inflammatory cell (lymphoplasmacytic) infiltration of the intestinal lamina propria accompanied by varying degrees of mucosal architectural disruption similar to that observed in tissue from human IBD patients (Figure 7).

**Figure 7.** Histopathological lesions of (A) crypt distortion with abscessation, (B) diffuse villous atrophy, and (C) mucos‐ al ulceration seen in duodenal biopsies of dogs with IBD.

Unfortunately, biopsy interpretation is notoriously subjective and suffers from extensive intraobserver variability and the technical constraints of procurement/processing artifacts inherent in evaluation of endoscopic specimens [236]. Although several histopathological scoring schemes have been proposed there are no uniform grading criteria that pathologists can universally agree on. One small study has resulted in development of a 'simplified model system' for defining intestinal inflammation of IBD that is presently being tested in a separate clinical trial.

#### *2.5.2. Therapeutic approach*

Treatment principles for canine IBD are empirical and consist of combination therapy us‐ ing both dietary and pharmacologic interventions. As compared to clinical trials evaluat‐ ing the efficacy of therapy for CD and UC, only one randomized, controlled drug trial for canine IBD has been reported [235]. There are, however, abundant evidence-based ob‐ servations that feeding elimination diets and administering corticosteroids, immunosup‐ pressive drugs, and/or select antibiotics are useful in the clinical management of canine IBD. Some clinicians prefer a sequential approach to nutritional and drug therapy for IBD. The optimal drug or drug combinations as well as duration of therapy for induc‐ tion and maintenance of remission of clinical signs have not been determined for most protocols [216, 230]. In general, the administration of corticosteroids (i.e., prednisone, prednisolone or budesonide), antimicrobials (i.e., metronidazole or tylosin), and immuno‐ suppressive drugs (i.e., cyclosporine, chlorambucil, azathioprine) used alone or in some combination are effective in inducing clinical remission in most animals. Some dogs will require intermittent or life-long drug therapy.

The rationale for nutritional therapy of IBD is that restricting exposure to antigens (i.e., dietary proteins) known to evoke sensitivity will reduce exaggerated host responses and attenuate intestinal inflammation. Other indications for specialized nutrition include the presence of decreased appetite, impaired nutrient absorption, or enteric plasma protein loss seen with moderate-to-severe mucosal inflammation. While evidence-based observations indicate that most dogs respond favorably to dietary intervention, the superiority of one novel protein source versus another or the advantage in feeding an intact protein elimination diet versus a hydrolyzed protein elimination diet has not been shown to date. Modifying the dietary n3:n6 fatty-acid ratio may also modulate inflammatory responses by reducing production of proinflammatory metabolites [237]. There is relatively sparse clinical data investigating prebiotic or probiotic therapy for canine IBD (see subsequent section on probiotics).

## *2.5.3. Future directions in canine IBD*

hypoadrenocorticism) causing chronic GI signs in dogs. The measure of clinical disease activity

A canine IBD activity index (CIBDAI) used for assessment of inflammatory activity in dogs has been recently designed [234]. Similar to other indices, the magnitude of the numerical score is proportional to the degree of inflammatory activity. This index serves as the principal measure of response to a therapeutic regimen and may be used to tailor medical therapy for an individual patient's needs [235]. Intestinal biopsies are required to confirm histopatholog‐ ical inflammation and to determine the extent of mucosal disease. Diagnostic endoscopy is preferred since this technique allows for direct assessment of mucosal abnormalities and the acquisition of targeted biopsy specimens. The microscopic findings in canine IBD consist of minimal to pronounced inflammatory cell (lymphoplasmacytic) infiltration of the intestinal lamina propria accompanied by varying degrees of mucosal architectural disruption similar

**Figure 7.** Histopathological lesions of (A) crypt distortion with abscessation, (B) diffuse villous atrophy, and (C) mucos‐

Unfortunately, biopsy interpretation is notoriously subjective and suffers from extensive intraobserver variability and the technical constraints of procurement/processing artifacts inherent in evaluation of endoscopic specimens [236]. Although several histopathological scoring schemes have been proposed there are no uniform grading criteria that pathologists can universally agree on. One small study has resulted in development of a 'simplified model system' for defining intestinal inflammation of IBD that is presently being tested in a separate

Treatment principles for canine IBD are empirical and consist of combination therapy us‐ ing both dietary and pharmacologic interventions. As compared to clinical trials evaluat‐ ing the efficacy of therapy for CD and UC, only one randomized, controlled drug trial for canine IBD has been reported [235]. There are, however, abundant evidence-based ob‐ servations that feeding elimination diets and administering corticosteroids, immunosup‐ pressive drugs, and/or select antibiotics are useful in the clinical management of canine IBD. Some clinicians prefer a sequential approach to nutritional and drug therapy for IBD. The optimal drug or drug combinations as well as duration of therapy for induc‐

by means of quantifiable indices is well established in human IBD [231-233].

to that observed in tissue from human IBD patients (Figure 7).

al ulceration seen in duodenal biopsies of dogs with IBD.

clinical trial.

96 Inflammatory Bowel Disease

*2.5.2. Therapeutic approach*

Canine IBD represents a common and frustrating GI disorder in veterinary medicine. More research is needed to unravel the mechanisms responsible for disease development and to translate these findings directly to human IBD. The primary features of IBD in humans and animals are remarkably similar (Table 1).

**Table 1.** Comparative features of IBD in humans and dogs.

Recent advances in clinical indices, histopathological standards, and the development of species-specific immunologic reagents and innovative molecular tools have made the dog an excellent 'spontaneous' animal model to study chronic immunologically-mediated intestinal inflammation. In addition, the dog has higher genomic sequence similarity to that of humans than do mice, a species traditionally used for comparative disease genetics [238]. However, clinical manifestations of complex disease in the mouse do not compare to the human form as closely as they do in the dog. Furthermore, the lifespan of the dog is much shorter than that of a human; thus, clinical trials aimed at treatment of IBD can be carried out much quicker and yield results that should have relevant application to human trials [230, 235, 239].

Clofazamine has also been documented to significantly affect the rate of remission in CD patients [241]. This drug, used to treat leprosy, is similar to ciprofloxacin, in that it has both anti-bacterial and anti-inflammatory properties [247-249]. It is only effective against Grampositive organisms, and its effectiveness increases in anaerobic environments [250]. Its effects on the immune system include increasing the presence of lysosomal enzymes in macrophages [251] and increasing phagocytosis by macrophages resulting in enhanced uptake and digestion of immune complexes [252]. Clofazamine has also been shown to inhibit TCR-mediated IL-2 production by T cells, thereby limiting T cell activation, a component of the pathogenesis of

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The most commonly used corticosteroids used are prednisolone, methylprednisolone, and budesonide [254]. These drugs are very effective at inducing remission, however, they are not without side effects. In a study comparing prednisolone and budesonide, both were found to be effective at inducing remission [255]. However, patients on budesonide had reduced evidence of adrenal axis suppression and peripheral leucocyte counts compared to those treated with prednisolone. These results indicate that budesonide is a safer choice yet it is still as effective as prednisolone. Corticosteroids are able to prevent NFκB activation [256] as well as block infiltration of neutrophils, prevent vasodilation and enhanced vascular permeability and downregulate the production of pro-inflammatory cytokines [254]. Although it is well established that these corticosteroids have an anti-inflammatory effect, little research has been conducted on the effects they have on the gut microbiota. A study by Swidsinski et al., mentioned previously, indicated that administration of cortisol increased the population of *F.*

Drugs such as methotrexate, 6-mercaptopurine (6-MP) and azathioprine (the prodrug of 6-MP) work by inhibiting the proliferation and activation of lymphocytes and decreasing the production of pro-inflammatory cytokines [254]. 6-mercaptopurine (6-MP) and azathioprine are purine antagonists and inhibit cellular metabolism by interfering with DNA replication [257, 258]. Methotrexate is a folic acid analog that inhibits DNA synthesis and, therefore, has an anti-proliferative effect [259]. Antibacterial effects, including growth inhibition of MAP,

Sulfasalazine was the first 5-ASA-type drug developed and is a combination of sulfapyridine (an antimicrobial) and salicyclic acid (an anti-inflammatory) to form a pro-drug. Upon entering the colon, this pro-drug is cleaved by colonic bacteria into the two separate molecules [260, 261]. Unfortunately, the frequency of gastrointestinal side effects was quite high due to the sulfapyridine moiety [262, 263]. It was later determined that the active moiety is 5-aminosali‐ cyclic acid (5-ASA, mesalazine) [264]. More recently formulations of this pro-drug have eliminated the sulfapyridine moiety and replaced it with either a second salicyclic acid moiety

IBD [253].

*2.6.2. Corticosteroids*

*prausnitzii* in a dose dependent manner [209].

have also been demonstrated for methotrexate and 6-MP [258].

*2.6.3. Immunosuppressive therapy*

*2.6.4. 5-aminosalicyclic acid (5-ASA)*

#### **2.6. Treatment for human IBD**

Treatments for human IBD patients typically involve the use of anti-inflammatory drugs, antibiotics, and there is a growing trend of probiotics and prebiotics being studied to determine their effects on disease activity. When patients fail to respond to treatment, the last and most drastic treatment option is surgery to resect sections of the inflamed bowel [240]. Most treatment options are primarily focused on reducing the inflammation associated with IBD. Some of these drugs also impact the microbiota and those will be discussed below.

#### *2.6.1. Antibiotics*

Given the importance of the microbiota in the pathogenesis of IBD, antibiotic therapy may seem an obvious treatment option. A meta-analysis by Khan et al. in 2011 reviewed random‐ ized controlled trials utilizing antibiotics in the treatment of IBD [241]. Rifamycin derivatives, ciprofloxacin, and clofazamine all induced remission in CD patients. Rifaximin (a rifamycin derivative) is effective against both Gram-negative and Gram-positive anaerobes and aerobes; it is also poorly absorbed after oral administration, resulting in little to no systemic side effects [242, 243]. Analysis of the effect of the drug using an *in vitro* continuous culture method with fecal samples from CD patients revealed increases in beneficial bacteria post-rifaximin administration [244]. Significant increases in *Bifidobacterium* spp., the *Atopobium* cluster, and *F. prausnitzii* were noted, as were increases in levels of lactate, acetate, and propionate as determined by 1 H-NMR spectroscopy [244].

Ciprofloxacin is a fluoroquinolone with broad-spectrum antibiotic activity [245]. In addition to having anti-bacterial properties, this drug has also been shown to have immunomodulatory properties as well [246]. In a TNBS mouse model of colitis, administration of ciprofloxacin ameliorated disease as compared to mice given ceftazidime (an antibacterial with a similar spectrum of activity compared to ciprofloxacin) [245]. Clinically, mice treated with ciproflox‐ acin did not lose weight and had reduced histopathological inflammatory scores associated with their colons. Ciprofloxacin treated mice also had reduced expression of IL-1β, IL-8, and TNF-α as measured from colonic homogenates as well as reduced expression of NF-κB. Since ceftazidime was less effective in ameliorating GIT inflammation, there appears to be additional benefits (i.e., anti-inflammatory) to the use of ciprofloxacin in addition to its spectrum of antimicrobial activity.

Clofazamine has also been documented to significantly affect the rate of remission in CD patients [241]. This drug, used to treat leprosy, is similar to ciprofloxacin, in that it has both anti-bacterial and anti-inflammatory properties [247-249]. It is only effective against Grampositive organisms, and its effectiveness increases in anaerobic environments [250]. Its effects on the immune system include increasing the presence of lysosomal enzymes in macrophages [251] and increasing phagocytosis by macrophages resulting in enhanced uptake and digestion of immune complexes [252]. Clofazamine has also been shown to inhibit TCR-mediated IL-2 production by T cells, thereby limiting T cell activation, a component of the pathogenesis of IBD [253].

## *2.6.2. Corticosteroids*

Recent advances in clinical indices, histopathological standards, and the development of species-specific immunologic reagents and innovative molecular tools have made the dog an excellent 'spontaneous' animal model to study chronic immunologically-mediated intestinal inflammation. In addition, the dog has higher genomic sequence similarity to that of humans than do mice, a species traditionally used for comparative disease genetics [238]. However, clinical manifestations of complex disease in the mouse do not compare to the human form as closely as they do in the dog. Furthermore, the lifespan of the dog is much shorter than that of a human; thus, clinical trials aimed at treatment of IBD can be carried out much quicker and

Treatments for human IBD patients typically involve the use of anti-inflammatory drugs, antibiotics, and there is a growing trend of probiotics and prebiotics being studied to determine their effects on disease activity. When patients fail to respond to treatment, the last and most drastic treatment option is surgery to resect sections of the inflamed bowel [240]. Most treatment options are primarily focused on reducing the inflammation associated with IBD.

Given the importance of the microbiota in the pathogenesis of IBD, antibiotic therapy may seem an obvious treatment option. A meta-analysis by Khan et al. in 2011 reviewed random‐ ized controlled trials utilizing antibiotics in the treatment of IBD [241]. Rifamycin derivatives, ciprofloxacin, and clofazamine all induced remission in CD patients. Rifaximin (a rifamycin derivative) is effective against both Gram-negative and Gram-positive anaerobes and aerobes; it is also poorly absorbed after oral administration, resulting in little to no systemic side effects [242, 243]. Analysis of the effect of the drug using an *in vitro* continuous culture method with fecal samples from CD patients revealed increases in beneficial bacteria post-rifaximin administration [244]. Significant increases in *Bifidobacterium* spp., the *Atopobium* cluster, and *F. prausnitzii* were noted, as were increases in levels of lactate, acetate, and propionate as

Ciprofloxacin is a fluoroquinolone with broad-spectrum antibiotic activity [245]. In addition to having anti-bacterial properties, this drug has also been shown to have immunomodulatory properties as well [246]. In a TNBS mouse model of colitis, administration of ciprofloxacin ameliorated disease as compared to mice given ceftazidime (an antibacterial with a similar spectrum of activity compared to ciprofloxacin) [245]. Clinically, mice treated with ciproflox‐ acin did not lose weight and had reduced histopathological inflammatory scores associated with their colons. Ciprofloxacin treated mice also had reduced expression of IL-1β, IL-8, and TNF-α as measured from colonic homogenates as well as reduced expression of NF-κB. Since ceftazidime was less effective in ameliorating GIT inflammation, there appears to be additional benefits (i.e., anti-inflammatory) to the use of ciprofloxacin in addition to its spectrum of

yield results that should have relevant application to human trials [230, 235, 239].

Some of these drugs also impact the microbiota and those will be discussed below.

H-NMR spectroscopy [244].

**2.6. Treatment for human IBD**

98 Inflammatory Bowel Disease

*2.6.1. Antibiotics*

determined by 1

antimicrobial activity.

The most commonly used corticosteroids used are prednisolone, methylprednisolone, and budesonide [254]. These drugs are very effective at inducing remission, however, they are not without side effects. In a study comparing prednisolone and budesonide, both were found to be effective at inducing remission [255]. However, patients on budesonide had reduced evidence of adrenal axis suppression and peripheral leucocyte counts compared to those treated with prednisolone. These results indicate that budesonide is a safer choice yet it is still as effective as prednisolone. Corticosteroids are able to prevent NFκB activation [256] as well as block infiltration of neutrophils, prevent vasodilation and enhanced vascular permeability and downregulate the production of pro-inflammatory cytokines [254]. Although it is well established that these corticosteroids have an anti-inflammatory effect, little research has been conducted on the effects they have on the gut microbiota. A study by Swidsinski et al., mentioned previously, indicated that administration of cortisol increased the population of *F. prausnitzii* in a dose dependent manner [209].

## *2.6.3. Immunosuppressive therapy*

Drugs such as methotrexate, 6-mercaptopurine (6-MP) and azathioprine (the prodrug of 6-MP) work by inhibiting the proliferation and activation of lymphocytes and decreasing the production of pro-inflammatory cytokines [254]. 6-mercaptopurine (6-MP) and azathioprine are purine antagonists and inhibit cellular metabolism by interfering with DNA replication [257, 258]. Methotrexate is a folic acid analog that inhibits DNA synthesis and, therefore, has an anti-proliferative effect [259]. Antibacterial effects, including growth inhibition of MAP, have also been demonstrated for methotrexate and 6-MP [258].

## *2.6.4. 5-aminosalicyclic acid (5-ASA)*

Sulfasalazine was the first 5-ASA-type drug developed and is a combination of sulfapyridine (an antimicrobial) and salicyclic acid (an anti-inflammatory) to form a pro-drug. Upon entering the colon, this pro-drug is cleaved by colonic bacteria into the two separate molecules [260, 261]. Unfortunately, the frequency of gastrointestinal side effects was quite high due to the sulfapyridine moiety [262, 263]. It was later determined that the active moiety is 5-aminosali‐ cyclic acid (5-ASA, mesalazine) [264]. More recently formulations of this pro-drug have eliminated the sulfapyridine moiety and replaced it with either a second salicyclic acid moiety (disodium azodisalicylate [265]) or an inert carrier (balsalazide [266]) thereby reducing the side effects.

Anti-TNF-α therapy works via multi-factorial mechanisms. First, it neutralizes TNF-α by blocking its ability to bind to TNF receptors, thus, inhibiting the pro-inflammatory response. Second, Anti-TNF-α binds to cell surface bound TNF-α on CD4+ T cells and macrophages, resulting in both complement- and antibody-dependent cell-mediated cytotoxicity [282]. All three monoclonal antibodies bind to and neutralize both soluble and membrane forms of TNFα [281]. Infliximab and Adalimumab both mediate complement- and antibody-dependent cellmediated cytotoxicity; however, Certolizumab only mediates complement-dependent cellular cytotoxicity as it lacks of an Fc region. Furthermore, Infliximab and Adalimumab induce apoptosis in peripheral blood lymphocytes and monocytes, as well as cause degranulation and loss of membrane integrity of PMNs. These activities were not induced with Certolizumab. Lastly, all three monoclonal antibodies inhibit the production of IL-1β after LPS stimulation in vitro, suggesting that there is a sequential production of pro-inflammatory cytokines induced by microbial components. Infliximab and Adalimumab both inhibit T cell prolifera‐ tion in mixed lymphocyte reactions in vitro, again suggesting that anti-TNF-α monoclonal antibodies ameliorate the inflammation associated with IBD via more than one mode of action [283]. The impact of these therapies on the microbiota, however, is not well studied. As previously mentioned, treatment with Infliximab resulted in increased levels of *F. prausnitzii* after administration [209]. As mentioned above, it is clear that the host inflammatory response often negatively impacts (i.e., shapes) the composition of the GIT microbiota, and that controlling mucosal inflammation benefits the health of the microbiota as well. Otherwise, there have been no specific studies performed to directly evaluate the role of anti-TNF-α

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An estimated 70 % of IBD patients have reported using complementary and alternative medicine (CAM) products at some time to treat their symptoms In a Canadian study of IBD patients, some of the most commonly used CAM treatments included massage therapy, chiropractic visits, probiotics, herbs, and fish oils [284]. A systematic review of the literature on the use of herbal medicines reveals some anti-inflammatory benefits associated with the administration of these herbs to both animals and humans [285]. It should be stressed that prior to utilizing any herbal remedy, which are potentially biologically active, patients need to consult their physician. This is especially important because the number two reason patients using CAM gave as to why they sought these products was that "natural therapy is safe"[286]. Some of the biological properties associated with CAM products include the reduction of proinflammatory cytokines, increased antioxidant production, inhibition of leukotriene B4,

Increasing evidence supports a potential therapeutic role for prebiotic and probiotic therapy in human IBD [287, 288]. If IBD in dogs is indeed driven by loss of tolerance to components of the intestinal microbiota as it is in humans, then prebiotics and probiotics may also prove beneficial as primary or adjunct therapies with diet and drugs. *Probiotics* are living microor‐ ganisms that, upon ingestion in sufficient numbers, impart health benefits beyond those of inherent basic nutrition [289]. Lactobacilli and Bifidobacterium have been the most commonly

decreased NF-κB activation, and inhibition of platelet activation [285].

therapies on the gut microbiota.

*2.6.6. Complementary and alternative therapies*

In addition to being an anti-inflammatory agent [267-270], 5-ASA also affects the gut micro‐ biota. As mentioned previously, patients taking mesalamine had a reduction in mucosaadherent bacteria [198, 271]. 5-ASA has also been shown to inhibit the growth of MAP [272] and *Bacteroides* spp. [273] and moderately inhibit the growth of *C. difficile* and *C. perfringens* in culture [273]. This drug also effects bacterial gene expression, as *Salmonella enterica* serovar Typhimurium incubated with 5-ASA had no change in growth but differentially expressed 110 genes [274]. Those genes characterized were found to be involved in invasion, metabolism, and antibiotic and stress resistance. *In vitro* assays revealed attenuation in the invasiveness of *S. enterica* serovar Typhimurium towards HeLa cells when pretreated with 5-ASA. These results indicate that in addition to inhibiting host-mediated inflammation, 5-ASA also has the potential to affect the intestinal microbiota.

#### *2.6.5. Anti-TNF monoclonal antibodies*

The induction of TNF-α is most often a downstream event following the interaction of phlogistic microbial components with toll-like receptors (TLRs) on host cells. The interest in TNF-α as a therapeutic target for IBD treatment began when the expression and secretion of this cytokine was found to be increased in IBD patients [275, 276]. For example, pediatric patients with either active UC or CD present with elevated levels of TNF-α in their stool [275]. Additionally, the incubation of GIT tissue sections in culture medium has demonstrated that significantly elevated levels of TNF-α are spontaneously secreted from inflamed tissue of both UC and CD patients when compared to the amounts produced by non-inflamed tissue and tissue from otherwise healthy individuals [276]. The predominant cell type producing the TNFα has been shown to be the macrophage [276]. Based on the central role TNF-α appears to play in the pathogenesis of IBD, there was interest in developing a therapeutic approach to control the harmful effects of this cytokine.

Clinically, the use of monoclonal antibodies to treat IBD patients began with Infliximab, an IgG1 murine-human chimeric monoclonal antibody specific for TNF-α, which was approved for human use in 1998 for CD [277, 278]. This monoclonal antibody consists of human constant regions and murine antigen binding regions [277]. These chimeric antibodies reduce the risk of immunoreactivity that occurs when murine antibodies are used. In addition to being less immunoreactive, this chimeric antibody had improved binding and neutralization character‐ istics for TNF-α than that of the original murine antibody [277]. Another monoclonal anti-TNF antibody, Adalimumab, is a fully humanized IgG1 antibody that avoids the induction of antispecies IgG that neutralize the effectiveness of the anti-TNF-α reagent [278, 279]. Lastly, Certolizumab is a monoclonal antibody fragment with a polyethylene glycol moiety (PEGy‐ lated) [278]. Certolizumab lacks the crystallizable fragment (Fc) portion of the immunoglobulin molecule and is an IgG4 isotype unlike Infliximab and Adalimumab, which are IgG1 antibodies [278, 280, 281]. In addition, the PEGylation increases the half-life of the antibody thereby reducing the frequency of administration.

Anti-TNF-α therapy works via multi-factorial mechanisms. First, it neutralizes TNF-α by blocking its ability to bind to TNF receptors, thus, inhibiting the pro-inflammatory response. Second, Anti-TNF-α binds to cell surface bound TNF-α on CD4+ T cells and macrophages, resulting in both complement- and antibody-dependent cell-mediated cytotoxicity [282]. All three monoclonal antibodies bind to and neutralize both soluble and membrane forms of TNFα [281]. Infliximab and Adalimumab both mediate complement- and antibody-dependent cellmediated cytotoxicity; however, Certolizumab only mediates complement-dependent cellular cytotoxicity as it lacks of an Fc region. Furthermore, Infliximab and Adalimumab induce apoptosis in peripheral blood lymphocytes and monocytes, as well as cause degranulation and loss of membrane integrity of PMNs. These activities were not induced with Certolizumab. Lastly, all three monoclonal antibodies inhibit the production of IL-1β after LPS stimulation in vitro, suggesting that there is a sequential production of pro-inflammatory cytokines induced by microbial components. Infliximab and Adalimumab both inhibit T cell prolifera‐ tion in mixed lymphocyte reactions in vitro, again suggesting that anti-TNF-α monoclonal antibodies ameliorate the inflammation associated with IBD via more than one mode of action [283]. The impact of these therapies on the microbiota, however, is not well studied. As previously mentioned, treatment with Infliximab resulted in increased levels of *F. prausnitzii* after administration [209]. As mentioned above, it is clear that the host inflammatory response often negatively impacts (i.e., shapes) the composition of the GIT microbiota, and that controlling mucosal inflammation benefits the health of the microbiota as well. Otherwise, there have been no specific studies performed to directly evaluate the role of anti-TNF-α therapies on the gut microbiota.

## *2.6.6. Complementary and alternative therapies*

(disodium azodisalicylate [265]) or an inert carrier (balsalazide [266]) thereby reducing the side

In addition to being an anti-inflammatory agent [267-270], 5-ASA also affects the gut micro‐ biota. As mentioned previously, patients taking mesalamine had a reduction in mucosaadherent bacteria [198, 271]. 5-ASA has also been shown to inhibit the growth of MAP [272] and *Bacteroides* spp. [273] and moderately inhibit the growth of *C. difficile* and *C. perfringens* in culture [273]. This drug also effects bacterial gene expression, as *Salmonella enterica* serovar Typhimurium incubated with 5-ASA had no change in growth but differentially expressed 110 genes [274]. Those genes characterized were found to be involved in invasion, metabolism, and antibiotic and stress resistance. *In vitro* assays revealed attenuation in the invasiveness of *S. enterica* serovar Typhimurium towards HeLa cells when pretreated with 5-ASA. These results indicate that in addition to inhibiting host-mediated inflammation, 5-ASA also has the

The induction of TNF-α is most often a downstream event following the interaction of phlogistic microbial components with toll-like receptors (TLRs) on host cells. The interest in TNF-α as a therapeutic target for IBD treatment began when the expression and secretion of this cytokine was found to be increased in IBD patients [275, 276]. For example, pediatric patients with either active UC or CD present with elevated levels of TNF-α in their stool [275]. Additionally, the incubation of GIT tissue sections in culture medium has demonstrated that significantly elevated levels of TNF-α are spontaneously secreted from inflamed tissue of both UC and CD patients when compared to the amounts produced by non-inflamed tissue and tissue from otherwise healthy individuals [276]. The predominant cell type producing the TNFα has been shown to be the macrophage [276]. Based on the central role TNF-α appears to play in the pathogenesis of IBD, there was interest in developing a therapeutic approach to control

Clinically, the use of monoclonal antibodies to treat IBD patients began with Infliximab, an IgG1 murine-human chimeric monoclonal antibody specific for TNF-α, which was approved for human use in 1998 for CD [277, 278]. This monoclonal antibody consists of human constant regions and murine antigen binding regions [277]. These chimeric antibodies reduce the risk of immunoreactivity that occurs when murine antibodies are used. In addition to being less immunoreactive, this chimeric antibody had improved binding and neutralization character‐ istics for TNF-α than that of the original murine antibody [277]. Another monoclonal anti-TNF antibody, Adalimumab, is a fully humanized IgG1 antibody that avoids the induction of antispecies IgG that neutralize the effectiveness of the anti-TNF-α reagent [278, 279]. Lastly, Certolizumab is a monoclonal antibody fragment with a polyethylene glycol moiety (PEGy‐ lated) [278]. Certolizumab lacks the crystallizable fragment (Fc) portion of the immunoglobulin molecule and is an IgG4 isotype unlike Infliximab and Adalimumab, which are IgG1 antibodies [278, 280, 281]. In addition, the PEGylation increases the half-life of the antibody thereby

effects.

100 Inflammatory Bowel Disease

potential to affect the intestinal microbiota.

*2.6.5. Anti-TNF monoclonal antibodies*

the harmful effects of this cytokine.

reducing the frequency of administration.

An estimated 70 % of IBD patients have reported using complementary and alternative medicine (CAM) products at some time to treat their symptoms In a Canadian study of IBD patients, some of the most commonly used CAM treatments included massage therapy, chiropractic visits, probiotics, herbs, and fish oils [284]. A systematic review of the literature on the use of herbal medicines reveals some anti-inflammatory benefits associated with the administration of these herbs to both animals and humans [285]. It should be stressed that prior to utilizing any herbal remedy, which are potentially biologically active, patients need to consult their physician. This is especially important because the number two reason patients using CAM gave as to why they sought these products was that "natural therapy is safe"[286]. Some of the biological properties associated with CAM products include the reduction of proinflammatory cytokines, increased antioxidant production, inhibition of leukotriene B4, decreased NF-κB activation, and inhibition of platelet activation [285].

Increasing evidence supports a potential therapeutic role for prebiotic and probiotic therapy in human IBD [287, 288]. If IBD in dogs is indeed driven by loss of tolerance to components of the intestinal microbiota as it is in humans, then prebiotics and probiotics may also prove beneficial as primary or adjunct therapies with diet and drugs. *Probiotics* are living microor‐ ganisms that, upon ingestion in sufficient numbers, impart health benefits beyond those of inherent basic nutrition [289]. Lactobacilli and Bifidobacterium have been the most commonly used human probiotics, but multi-strain cocktails (e.g., VSL#3), *E. coli* Nissle 1917, and nonbacterial *Saccharomyces boulardii* have also been used as probiotics [226]. Probiotic bacteria have measurable host benefits, including the ability to improve epithelial barrier function, modulate the mucosal immune system, and alter the intestinal flora [290]. *Prebiotics* are nondigestible dietary carbohydrates, such as lactosucrose, fructo-oligosaccharides (FOS), psylli‐ um, bran, which beneficially stimulate the growth and metabolism of endogenous enteric bacteria upon consumption [291]. Beneficial effects of prebiotics are also associated with the production of short chain fatty acids (SCFA) due to fermentation by colonic bacteria. *Synbiot‐ ics* are combinations of probiotics and prebiotics that are an emerging therapeutic modality. Increasing evidence supports a therapeutic role for probiotics, prebiotics, and synbiotics in treating gastrointestinal diseases of humans, including infectious diarrhea, *H. pylori* infection, irritable bowel syndrome, lactase deficiency, and IBD [84]. A comparison of prebiotic and probiotic preparations is outlined in Table 2.

pro-inflammatory chemokine IP-10 [300]. The most recent studies with VSL#3 treatment of UC show increased fecal concentrations of beneficial bacterial species, improved clinical, endo‐ scopic and histopathological scores in most patients, and higher rates of remission compared to placebo [298]. The most recent studies with VSL#3 treatment of UC show increased fecal concentrations of beneficial bacterial species, improved clinical, endoscopic and histopatho‐ logical scores in most patients, and higher rates of remission compared to placebo [298]. VSL#3 is available without prescription and can be ordered via the internet or obtained locally at the

**Probiotics Prebiotics**

Non-digestible carbohydrate which stimulate replication of protective enteric bacteria when consumed

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Fructo-oligosaccharide (FOS) Galacto-oligosaccharide (GOS) Inulin Lactulose Psyllium Bran Beet pulp, pumpkin Resistant starch

Stimulates replication of beneficial bacteria (*Bifidobacterium*) Enhances production of SCFA (butyrate) Improved intestinal barrier function Decreases proinflammatory cytokines

pharmacy in the U.S. [301].

Definition Live microorganisms which, given in

Examples *E. coli* Nissle 1917

Protective Mechanisms Alters microbiota to suppress pathogens

**Table 2.** Basic Features of Probiotics and Prebiotics

adequate amounts confer health benefits to the host

> VSL#3 *Lactobacillus* species *Bifidobacterium* species Saccharomyces boulardii Prostora Max® Forti Flora® Proviable-DC®

Improved intestinal barrier function Increased production of antimicrobial peptides Decreased expression of proinflammatory cytokines

Recent studies have also shown that dogs with IBD have distinctly different duodenal microbial communities compared to healthy dogs. Current treatments for IBD include the administration of nonspecific anti-inflammatory drugs which may confer serious side effects and do not address the underlying basis for disease, namely, altered microbial composition. The use of probiotics offers an attractive, physiologic, and non-toxic alternative to shift the balance to protective species and treat canine IBD. The authors (AEJ) have initiated a clinical trial to investigate the clinical, microbiologic, and anti-inflammatory effects of probiotic VSL#3 in the treatment of canine IBD. We hypothesize that VSL#3 used as an adjunct to standard therapy (i.e., elimination diet and prednisone) will induce a beneficial alteration of enteric

#### *2.6.7. Probiotics*

VSL#3 is one of the most commonly used probiotic cocktails and contains a very high bacterial concentration per gram of product characterized by greater number of different bacterial species as compared to traditional probiotic preparations [292]. This commercially prepared formulation consists of 450 billion bacteria/g of viable lyophilized bacteria comprised of eight bacterial strains (*Lactobacillus casei, L plantarum, L. bulgaricus, L. acidolphilus, Bifidobacterium longum, B. breve, B. infantis* and *Streptococcus thermophiles*). While the exact mechanism of action of VSL#3 is unknown, several studies have demonstrated the effects of VSL#3 on epithelial barrier function and down regulation of cytokine secretion from immune cells. For example, Madsen [293] has shown in *in vitro* studies that epithelial barrier function could be enhanced by exposure to a soluble factor secreted by VSL#3 bacteria. Moreover, this same study demonstrated that VSL#3 did not alter the ability of the epithelial cell to activate a mucosal inflammatory response to a bacterial invasion. Studies with VSL#3 formulations have also been conducted in several animal models of colitis, inflammatory liver disease, sepsis, and irritable bowel syndrome (IBS). In models of experimentally-induced colitis, these studies demonstrat‐ ed that VSL#3 normalized gut permeability and barrier function, and that VSL#3 modulated inflammatory and immune responses [226, 294]. Animal models of sepsis have also demon‐ strated that VSL#3 administration reduced bacterial translocation and significantly attenuated damage to the liver and intestinal mucosa [295]. The use of VSL#3 as an innovative probiotic preparation, developed specifically to balance the intestinal microbiota, is supported by studies in humans with IBD (ulcerative colitis [296], pouchitis) and in other patients with diverse gastrointestinal disorders, such as IBS [297, 298].

Studies have shown VSL#3 to induce remission of inflammation in 77 % of adult UC patients with no adverse effects [298] and 56 % of pediatric UC patients [299]. These same pediatric UC patients had a reduction in disease activity index and sigmoidoscopy scores following VSL#3 treatment. They also had reduced levels of the pro-inflammatory cytokines TNF-α and IFNγ following therapy [299]. *In vitro* analysis of the effects of VSL#3 on Mode-K epithelial cells revealed that only one of the eight organisms, *L. casei*, inhibited TNF-induced secretion of the pro-inflammatory chemokine IP-10 [300]. The most recent studies with VSL#3 treatment of UC show increased fecal concentrations of beneficial bacterial species, improved clinical, endo‐ scopic and histopathological scores in most patients, and higher rates of remission compared to placebo [298]. The most recent studies with VSL#3 treatment of UC show increased fecal concentrations of beneficial bacterial species, improved clinical, endoscopic and histopatho‐ logical scores in most patients, and higher rates of remission compared to placebo [298]. VSL#3 is available without prescription and can be ordered via the internet or obtained locally at the pharmacy in the U.S. [301].

#### **Table 2.** Basic Features of Probiotics and Prebiotics

used human probiotics, but multi-strain cocktails (e.g., VSL#3), *E. coli* Nissle 1917, and nonbacterial *Saccharomyces boulardii* have also been used as probiotics [226]. Probiotic bacteria have measurable host benefits, including the ability to improve epithelial barrier function, modulate the mucosal immune system, and alter the intestinal flora [290]. *Prebiotics* are nondigestible dietary carbohydrates, such as lactosucrose, fructo-oligosaccharides (FOS), psylli‐ um, bran, which beneficially stimulate the growth and metabolism of endogenous enteric bacteria upon consumption [291]. Beneficial effects of prebiotics are also associated with the production of short chain fatty acids (SCFA) due to fermentation by colonic bacteria. *Synbiot‐ ics* are combinations of probiotics and prebiotics that are an emerging therapeutic modality. Increasing evidence supports a therapeutic role for probiotics, prebiotics, and synbiotics in treating gastrointestinal diseases of humans, including infectious diarrhea, *H. pylori* infection, irritable bowel syndrome, lactase deficiency, and IBD [84]. A comparison of prebiotic and

VSL#3 is one of the most commonly used probiotic cocktails and contains a very high bacterial concentration per gram of product characterized by greater number of different bacterial species as compared to traditional probiotic preparations [292]. This commercially prepared formulation consists of 450 billion bacteria/g of viable lyophilized bacteria comprised of eight bacterial strains (*Lactobacillus casei, L plantarum, L. bulgaricus, L. acidolphilus, Bifidobacterium longum, B. breve, B. infantis* and *Streptococcus thermophiles*). While the exact mechanism of action of VSL#3 is unknown, several studies have demonstrated the effects of VSL#3 on epithelial barrier function and down regulation of cytokine secretion from immune cells. For example, Madsen [293] has shown in *in vitro* studies that epithelial barrier function could be enhanced by exposure to a soluble factor secreted by VSL#3 bacteria. Moreover, this same study demonstrated that VSL#3 did not alter the ability of the epithelial cell to activate a mucosal inflammatory response to a bacterial invasion. Studies with VSL#3 formulations have also been conducted in several animal models of colitis, inflammatory liver disease, sepsis, and irritable bowel syndrome (IBS). In models of experimentally-induced colitis, these studies demonstrat‐ ed that VSL#3 normalized gut permeability and barrier function, and that VSL#3 modulated inflammatory and immune responses [226, 294]. Animal models of sepsis have also demon‐ strated that VSL#3 administration reduced bacterial translocation and significantly attenuated damage to the liver and intestinal mucosa [295]. The use of VSL#3 as an innovative probiotic preparation, developed specifically to balance the intestinal microbiota, is supported by studies in humans with IBD (ulcerative colitis [296], pouchitis) and in other patients with

Studies have shown VSL#3 to induce remission of inflammation in 77 % of adult UC patients with no adverse effects [298] and 56 % of pediatric UC patients [299]. These same pediatric UC patients had a reduction in disease activity index and sigmoidoscopy scores following VSL#3 treatment. They also had reduced levels of the pro-inflammatory cytokines TNF-α and IFNγ following therapy [299]. *In vitro* analysis of the effects of VSL#3 on Mode-K epithelial cells revealed that only one of the eight organisms, *L. casei*, inhibited TNF-induced secretion of the

probiotic preparations is outlined in Table 2.

diverse gastrointestinal disorders, such as IBS [297, 298].

*2.6.7. Probiotics*

102 Inflammatory Bowel Disease

Recent studies have also shown that dogs with IBD have distinctly different duodenal microbial communities compared to healthy dogs. Current treatments for IBD include the administration of nonspecific anti-inflammatory drugs which may confer serious side effects and do not address the underlying basis for disease, namely, altered microbial composition. The use of probiotics offers an attractive, physiologic, and non-toxic alternative to shift the balance to protective species and treat canine IBD. The authors (AEJ) have initiated a clinical trial to investigate the clinical, microbiologic, and anti-inflammatory effects of probiotic VSL#3 in the treatment of canine IBD. We hypothesize that VSL#3 used as an adjunct to standard therapy (i.e., elimination diet and prednisone) will induce a beneficial alteration of enteric bacteria leading to induction and maintenance of remission in dogs with IBD. A randomized, controlled clinical trial of eight weeks duration will assess the efficacy of standard therapy in conjunction with VSL#3 versus standard therapy alone in the management of canine IBD. There is a need for additional data to be generated to provide proof of efficacy in probiotic therapy before these agents can be applied to widespread clinical use. These studies will also provide highly relevant insight into the anti-inflammatory effects of probiotics for treatment of human and canine IBD.

and *F. prausnitzii* also has other anti-inflammatory properties. In animal models, prebiot‐ ics have been shown to be effective as well. The severity of DSS-induced colitis in rats was attenuated by administering inulin orally as evidenced by a reduction in histopatho‐ logical scores and myeloperioxidase accumulation in the colons [315]. In addition, the acidity of the colonic contents increased in rats fed inulin as well as an increase in *Lacto‐ bacillus* spp. in the feces. In a multicenter trial feeding UC patients germinated barley foodstuff (GBF), patients consuming GBF had improved clinical activity index scores [316]. GBF has also been shown to be effective in maintaining remission [317] and reduc‐ ing pro-inflammatory cytokine levels in UC patients [318]. In CD patients, dietary sup‐ plementation with fructo-oligosaccharide (FOS) reduced their disease activity index, increased fecal *Bifidobacterium* spp. levels as well as increased production of IL-10 and ex‐

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Scientific studies have also investigated the effects of dietary supplementation with prebiotics on the intestinal microbiota of healthy dogs and cats. In one study, FOS supplemented at 0.75 % dry matter produced qualitative and quantitative changes in the fecal flora of healthy cats [320]. Compared with samples from cats fed a basal diet, increased numbers of lactobacilli and *Bacteroides* spp. and decreased numbers of *E. coli* were associated with cats fed the FOS supplemented diet. However, bacteriologic examination of the duodenal juice in these same cats showed wide variation in the composition of the duodenal microbiota, across sampling periods, which was not affected by FOS supplementation [321]. Moreover, healthy Beagle dogs fed a 1 % FOS diet over a three-month trial showed inconsistent fecal excretion of *Lactobacil‐ lus* spp. and *Bifidobacterium* spp. [322]. While FOS supplementation has been shown to have health benefits, these studies demonstrate that FOS does not have an adverse affect on the microbiota and suggest that it may have positive physiological benefits as seen in humans. This observation and the lack of significant side effects associated with FOS supplementation provide evidence that FOS should be considered as an attractive alternative or adjunct therapy

Many different mouse models have been utilized in IBD research to elucidate the roles that bacteria, genetics, the immune response, and environment play in the induction and mainte‐ nance of IBD [323]. They fall into two main categories: chemically-induced and geneticallyengineered models. The composition of the microbiota in the various mouse models is also discussed. Regardless of the strain of mouse employed in chemically-induced or geneticallyengineered models, there are only five options for its microbiota—conventional, specific pathogen free, restricted, gnotobiotic, or germ-free. Note that the word gnotobiotic (gnostos– "known" bios –"life") indicates that all the organisms present, regardless of the numbers of species, are known and does not apply only to mice that are completely devoid of microbes

pression of TLR-2 and TLR-4 from lamina propria dendritic cells [319].

for IBD in dogs and cats.

(i.e., germfree).

**3. Mouse models of IBD**

Another popular probiotic is *E. coli* Nissle 1917 also known as Mutaflor®. This probiotic has been shown to be just as effective as mesalazine in achieving and maintaining remission of inflammation in UC patients [302-304]. It has recently been shown that when *E. coli* Nissle 1917 is genetically modified to produce the quorum-sensing molecule AI-2, it affected the beneficial probiotic properties of this organism [305]. Colonization of healthy newborn infants with *E. coli* Nissle 1917 has shown that the presence of pathogenic bacteria in the gut was significantly reduced compared to non-colonized infants [306]. *In vitro* studies have shown *E. coli* Nissle 1917 is able to reduce the invasive ability of multiple pathogenic organisms [307], and also to reduce the adherent and invasive ability of AIEC [308]. *E. coli* Nissle 1917 was also shown to either inhibit the growth (49 %) or overgrowth (30 %) of 79 % of uropathogenic organisms recovered from children with urinary tract infections [309]. Unlike VSL#3, Mutaflor® cannot be purchased in the U.S. due to its reclassification by the F.D.A. from a "medical food" (which is what VSL#3 is classified as) to a "biologic" [301, 310].

There are few reports on the use of probiotic bacteria in dogs and cats. Recent *in vitro* studies have confirmed the capacity of a lyophilized probiotic cocktail (e.g., three different *Lactobacil‐ lus* spp strains) to modulate the expression of regulatory versus pro-inflammatory cytokines in dogs with chronic enteropathies [311]. However, a clinical trial using this same probiotic cocktail fed to dogs with food-responsive diarrhea failed to induce consistent patterns of regulatory (e.g., beneficial) cytokine expression in spite of obvious clinical improvement [312]. One commercially-manufactured probiotic (FortiFlora™–*Enterococcus faecium* SF68, Nestle Purina) is reported to potentially control diarrhea and enhance immune responses in dogs and cats. Several recent trials attest to the short-term efficacy of probiotics in treating acute diarrhea in dogs and cats [313]. The link between the intestinal microbiota and gastrointestinal health in companion animals is now obvious. Future developments in the pharmabiotic field must include performance of randomized clinical trials to determine the role of probiotics and prebiotics in the management of canine chronic enteropathy. One large multicenter trial investigating the efficacy of VSL#3 in reducing inflammatory activity of canine IBD is presently underway

#### *2.6.8. Prebiotics*

Prebiotics are substances that can be used to promote specific changes in the microbiota. Administration has been shown to shift the microbiota in healthy adults; for example, in‐ dividuals who consume either soluble corn fiber or polydextrose had increases in *F. prausnitzii* and those who consumed the soluble corn fiber had increases in *Roseburia* spp. [314]. As mentioned previously, both of these organisms metabolically produce butyrate and *F. prausnitzii* also has other anti-inflammatory properties. In animal models, prebiot‐ ics have been shown to be effective as well. The severity of DSS-induced colitis in rats was attenuated by administering inulin orally as evidenced by a reduction in histopatho‐ logical scores and myeloperioxidase accumulation in the colons [315]. In addition, the acidity of the colonic contents increased in rats fed inulin as well as an increase in *Lacto‐ bacillus* spp. in the feces. In a multicenter trial feeding UC patients germinated barley foodstuff (GBF), patients consuming GBF had improved clinical activity index scores [316]. GBF has also been shown to be effective in maintaining remission [317] and reduc‐ ing pro-inflammatory cytokine levels in UC patients [318]. In CD patients, dietary sup‐ plementation with fructo-oligosaccharide (FOS) reduced their disease activity index, increased fecal *Bifidobacterium* spp. levels as well as increased production of IL-10 and ex‐ pression of TLR-2 and TLR-4 from lamina propria dendritic cells [319].

Scientific studies have also investigated the effects of dietary supplementation with prebiotics on the intestinal microbiota of healthy dogs and cats. In one study, FOS supplemented at 0.75 % dry matter produced qualitative and quantitative changes in the fecal flora of healthy cats [320]. Compared with samples from cats fed a basal diet, increased numbers of lactobacilli and *Bacteroides* spp. and decreased numbers of *E. coli* were associated with cats fed the FOS supplemented diet. However, bacteriologic examination of the duodenal juice in these same cats showed wide variation in the composition of the duodenal microbiota, across sampling periods, which was not affected by FOS supplementation [321]. Moreover, healthy Beagle dogs fed a 1 % FOS diet over a three-month trial showed inconsistent fecal excretion of *Lactobacil‐ lus* spp. and *Bifidobacterium* spp. [322]. While FOS supplementation has been shown to have health benefits, these studies demonstrate that FOS does not have an adverse affect on the microbiota and suggest that it may have positive physiological benefits as seen in humans. This observation and the lack of significant side effects associated with FOS supplementation provide evidence that FOS should be considered as an attractive alternative or adjunct therapy for IBD in dogs and cats.

## **3. Mouse models of IBD**

bacteria leading to induction and maintenance of remission in dogs with IBD. A randomized, controlled clinical trial of eight weeks duration will assess the efficacy of standard therapy in conjunction with VSL#3 versus standard therapy alone in the management of canine IBD. There is a need for additional data to be generated to provide proof of efficacy in probiotic therapy before these agents can be applied to widespread clinical use. These studies will also provide highly relevant insight into the anti-inflammatory effects of probiotics for treatment

Another popular probiotic is *E. coli* Nissle 1917 also known as Mutaflor®. This probiotic has been shown to be just as effective as mesalazine in achieving and maintaining remission of inflammation in UC patients [302-304]. It has recently been shown that when *E. coli* Nissle 1917 is genetically modified to produce the quorum-sensing molecule AI-2, it affected the beneficial probiotic properties of this organism [305]. Colonization of healthy newborn infants with *E. coli* Nissle 1917 has shown that the presence of pathogenic bacteria in the gut was significantly reduced compared to non-colonized infants [306]. *In vitro* studies have shown *E. coli* Nissle 1917 is able to reduce the invasive ability of multiple pathogenic organisms [307], and also to reduce the adherent and invasive ability of AIEC [308]. *E. coli* Nissle 1917 was also shown to either inhibit the growth (49 %) or overgrowth (30 %) of 79 % of uropathogenic organisms recovered from children with urinary tract infections [309]. Unlike VSL#3, Mutaflor® cannot be purchased in the U.S. due to its reclassification by the F.D.A. from a "medical food" (which

There are few reports on the use of probiotic bacteria in dogs and cats. Recent *in vitro* studies have confirmed the capacity of a lyophilized probiotic cocktail (e.g., three different *Lactobacil‐ lus* spp strains) to modulate the expression of regulatory versus pro-inflammatory cytokines in dogs with chronic enteropathies [311]. However, a clinical trial using this same probiotic cocktail fed to dogs with food-responsive diarrhea failed to induce consistent patterns of regulatory (e.g., beneficial) cytokine expression in spite of obvious clinical improvement [312]. One commercially-manufactured probiotic (FortiFlora™–*Enterococcus faecium* SF68, Nestle Purina) is reported to potentially control diarrhea and enhance immune responses in dogs and cats. Several recent trials attest to the short-term efficacy of probiotics in treating acute diarrhea in dogs and cats [313]. The link between the intestinal microbiota and gastrointestinal health in companion animals is now obvious. Future developments in the pharmabiotic field must include performance of randomized clinical trials to determine the role of probiotics and prebiotics in the management of canine chronic enteropathy. One large multicenter trial investigating the efficacy of VSL#3 in reducing inflammatory activity of canine IBD is presently

Prebiotics are substances that can be used to promote specific changes in the microbiota. Administration has been shown to shift the microbiota in healthy adults; for example, in‐ dividuals who consume either soluble corn fiber or polydextrose had increases in *F. prausnitzii* and those who consumed the soluble corn fiber had increases in *Roseburia* spp. [314]. As mentioned previously, both of these organisms metabolically produce butyrate

of human and canine IBD.

104 Inflammatory Bowel Disease

underway

*2.6.8. Prebiotics*

is what VSL#3 is classified as) to a "biologic" [301, 310].

Many different mouse models have been utilized in IBD research to elucidate the roles that bacteria, genetics, the immune response, and environment play in the induction and mainte‐ nance of IBD [323]. They fall into two main categories: chemically-induced and geneticallyengineered models. The composition of the microbiota in the various mouse models is also discussed. Regardless of the strain of mouse employed in chemically-induced or geneticallyengineered models, there are only five options for its microbiota—conventional, specific pathogen free, restricted, gnotobiotic, or germ-free. Note that the word gnotobiotic (gnostos– "known" bios –"life") indicates that all the organisms present, regardless of the numbers of species, are known and does not apply only to mice that are completely devoid of microbes (i.e., germfree).

## **3.1. Chemically-induced models**

#### *3.1.1. DSS*

Dextran sulfate sodium (DSS) is formed by the esterification of dextran with chlorosulphonic acid [324]. Administered *ad libtum* in the drinking water, this compound causes enterocolitis in mice with disease severity being dependent on mouse strain, DSS molecular mass, and sulfur content [325-328]. Within three to seven days after the addition of DSS (1 to 10 % w/v) to the drinking water, mice exhibit loose stools, weight loss, and occult blood. Upon necropsy of mice treated with DSS, cecal atrophy and colonic shortening are noted, and histopathological examination reveals mucosal ulceration, inflammatory infiltrate, and hyperplastic epithelium in the colonic mucosa. It has also been reported that glandular dropout occurs prior to signs of inflammation. Although the exact mechanism of action for DSS-induced colitis is currently not known, it is thought to manifest epithelial toxicity [329, 330]. Other reports indicate that DSS increases mucosal permeability within three days of administration (before the appear‐ ance of inflammatory infiltrate) [331]. Studies have also shown that DSS is taken up by macrophages in the colon and mesenteric lymph node and by Kupffer cells in the liver [326, 330]. When macrophages become laden with DSS, they have a reduced ability to perform normal homeostatic functions such as tissue repair and phagocytosis of bacteria [332]. DSS was demonstrated to be cytotoxic to Caco-2 cells, binding to their nucleus, causing cell cycle arrest and reduced production of reactive oxygen species [333].

inflammatory cell infiltrate that produced IFN-γ [336]. In that same study, researchers found that feeding mice TNBS-haptenized colonic protein caused the mice to develop or‐ al tolerance. These mice subsequently failed to develop colitis after TNBS administration or the transfer of CD4<sup>+</sup> T cells from mice with TNBS-induced colitis. T cells from these

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Oxazolone is a haptenating agent that causes a disease in mice similar to UC when mixed with ethanol and administered rectally [337, 338]. SJL/J mice rapidly develop diarrhea and weight loss that peaks at day two post-administration with a 50 % mortality rate by day four. At day two, the distal half of the colon becomes hemorrhagic and edematous and histologically shows signs of superficial inflammation. There is epithelial cell erosion, gob‐ let cell depletion, edema, and inflammatory cell infiltrate composed of neutrophils and eo‐ sinophils. This is similar to what is observed microscopically in the colonic tissues of human UC patients. These mice also develop elevated levels of IL-4 and IL-5, but no IFNγ, indicating that oxazolone induces a Th2 response. Elevated levels of TGF-β are also noted and may play in a role in the induction of disease in only part of the colon. The model has also been examined for its role in determining efficacy of IBD treatments. BALB/c mice given either 5-aminosalicylic acid (5-ASA) or sodium prednisolone phos‐ phate intra-rectally prior to and during induction of oxazolone colitis had decreased se‐ verity of disease [337]. The disease is self-limiting, and the mice that survive beyond day four show increased weight gain and are healthy by days 10-12 post-administration.

In 1993, Kuhn et al. discovered that mice deficient in the anti-inflammatory cytokine IL-10 spontaneously develop enterocolitis [213]. This model has been popular in IBD research and this genetic deficiency has been crossed onto many different genetic backgrounds of mouse [339-345]. The availability of different strains has highlighted the role that genetics plays in enterocolitis, as the severity of disease is strain dependent. The order of severity from most severe to least severe is as follows: C3Bir > 129 > BALB/c or NOD/Lt > C57BL/ 6 or C57BL/10. In addition to strain differences, development of entercolitis is also de‐ pendent on the microbiota, as GF IL-10-/- do not develop enterocolitis and disease is atte‐ nuated after administration of antibiotics to IL-10-/- mice harboring a conventional microbiota [346, 347]. The lack of IL-10 does not affect the development of B or T cells, but its absence does result in a lack of regulatory T cells [213, 348]. Studies to under‐ stand the development of disease in these mice have shown that B cells (while present in high numbers in the lamina propria) are not needed for the initiation of disease, but that disease is mediated by CD4<sup>+</sup> T cells [342, 349]. Transfer studies using RAG2-/- mice as re‐ cipients have specifically shown that naive CD4<sup>+</sup> T cells are capable of inducing colitis, and CD45RBlow CD4+ T cells from IL-10-/- mice can induce disease in these RAG2-/- mice

tolerant mice secreted elevated amounts of TGF-β, IL-4, and IL-10 [336].

*3.1.3. Oxazolone*

**3.2. Genetically engineered models**

*3.2.1. IL-10-/-*

Microbial populations of the GIT are altered after DSS administration, with the microbiota of the treated mice having increased numbers of *Bacteroidaceae* and *Clostridium* spp. [326]. This result indicates that the changes to the microbiota may play a contributory role (e.g., reduction of butyrate production) in the induction of DSS-induced disease. However, DSS induces more severe colitis and increased mortality in GF mice as compared to their conventional counter‐ parts [334, 335]. GF mice given either 5% or 1% DSS died at days three or 14, respectively, after the start of DSS administration [335]. Collectively, these studies indicate that the composition of the microbiota may influence the sensitivity of mice to DSS-induced colitis as well as support the idea that the resident microbiota affords cytoprotective benefits for the host.

#### *3.1.2. TNBS (2,4,6,-trinitrobenzne sulfonic acid)*

TNBS is a haptenating agent that causes a disease similar to CD when mixed with etha‐ nol and administered rectally as an enema. Mice treated with TNBS develop a pan-colitis with the peak of clinical signs, such as diarrhea and rectal prolapse, occurring two to four weeks post-administration [112]. Microscopically, transmural inflammation is noted along with neutrophil infiltration, loss of goblet cells, edema, and granulomas. T cells isolated from the lamina propria secrete elevated levels of IFN-γ and IL-2 following stimulation with anti-CD3 and anti-CD28. However, administration of anti-IL-12 antibod‐ ies after induction of TNBS-induced colitis reduced the disease severity, and treated mice also showed reduced production of IFN-γ [112]. Further studies have shown that CD4+ T cells recovered from mice with TNBS-induced colitis could induce mild colitis when adoptively transferred into naive control mice; the colitic lesions were characterized by inflammatory cell infiltrate that produced IFN-γ [336]. In that same study, researchers found that feeding mice TNBS-haptenized colonic protein caused the mice to develop or‐ al tolerance. These mice subsequently failed to develop colitis after TNBS administration or the transfer of CD4<sup>+</sup> T cells from mice with TNBS-induced colitis. T cells from these tolerant mice secreted elevated amounts of TGF-β, IL-4, and IL-10 [336].

### *3.1.3. Oxazolone*

**3.1. Chemically-induced models**

Dextran sulfate sodium (DSS) is formed by the esterification of dextran with chlorosulphonic acid [324]. Administered *ad libtum* in the drinking water, this compound causes enterocolitis in mice with disease severity being dependent on mouse strain, DSS molecular mass, and sulfur content [325-328]. Within three to seven days after the addition of DSS (1 to 10 % w/v) to the drinking water, mice exhibit loose stools, weight loss, and occult blood. Upon necropsy of mice treated with DSS, cecal atrophy and colonic shortening are noted, and histopathological examination reveals mucosal ulceration, inflammatory infiltrate, and hyperplastic epithelium in the colonic mucosa. It has also been reported that glandular dropout occurs prior to signs of inflammation. Although the exact mechanism of action for DSS-induced colitis is currently not known, it is thought to manifest epithelial toxicity [329, 330]. Other reports indicate that DSS increases mucosal permeability within three days of administration (before the appear‐ ance of inflammatory infiltrate) [331]. Studies have also shown that DSS is taken up by macrophages in the colon and mesenteric lymph node and by Kupffer cells in the liver [326, 330]. When macrophages become laden with DSS, they have a reduced ability to perform normal homeostatic functions such as tissue repair and phagocytosis of bacteria [332]. DSS was demonstrated to be cytotoxic to Caco-2 cells, binding to their nucleus, causing cell cycle

Microbial populations of the GIT are altered after DSS administration, with the microbiota of the treated mice having increased numbers of *Bacteroidaceae* and *Clostridium* spp. [326]. This result indicates that the changes to the microbiota may play a contributory role (e.g., reduction of butyrate production) in the induction of DSS-induced disease. However, DSS induces more severe colitis and increased mortality in GF mice as compared to their conventional counter‐ parts [334, 335]. GF mice given either 5% or 1% DSS died at days three or 14, respectively, after the start of DSS administration [335]. Collectively, these studies indicate that the composition of the microbiota may influence the sensitivity of mice to DSS-induced colitis as well as support

TNBS is a haptenating agent that causes a disease similar to CD when mixed with etha‐ nol and administered rectally as an enema. Mice treated with TNBS develop a pan-colitis with the peak of clinical signs, such as diarrhea and rectal prolapse, occurring two to four weeks post-administration [112]. Microscopically, transmural inflammation is noted along with neutrophil infiltration, loss of goblet cells, edema, and granulomas. T cells isolated from the lamina propria secrete elevated levels of IFN-γ and IL-2 following stimulation with anti-CD3 and anti-CD28. However, administration of anti-IL-12 antibod‐ ies after induction of TNBS-induced colitis reduced the disease severity, and treated mice also showed reduced production of IFN-γ [112]. Further studies have shown that CD4+ T cells recovered from mice with TNBS-induced colitis could induce mild colitis when adoptively transferred into naive control mice; the colitic lesions were characterized by

the idea that the resident microbiota affords cytoprotective benefits for the host.

arrest and reduced production of reactive oxygen species [333].

*3.1.2. TNBS (2,4,6,-trinitrobenzne sulfonic acid)*

*3.1.1. DSS*

106 Inflammatory Bowel Disease

Oxazolone is a haptenating agent that causes a disease in mice similar to UC when mixed with ethanol and administered rectally [337, 338]. SJL/J mice rapidly develop diarrhea and weight loss that peaks at day two post-administration with a 50 % mortality rate by day four. At day two, the distal half of the colon becomes hemorrhagic and edematous and histologically shows signs of superficial inflammation. There is epithelial cell erosion, gob‐ let cell depletion, edema, and inflammatory cell infiltrate composed of neutrophils and eo‐ sinophils. This is similar to what is observed microscopically in the colonic tissues of human UC patients. These mice also develop elevated levels of IL-4 and IL-5, but no IFNγ, indicating that oxazolone induces a Th2 response. Elevated levels of TGF-β are also noted and may play in a role in the induction of disease in only part of the colon. The model has also been examined for its role in determining efficacy of IBD treatments. BALB/c mice given either 5-aminosalicylic acid (5-ASA) or sodium prednisolone phos‐ phate intra-rectally prior to and during induction of oxazolone colitis had decreased se‐ verity of disease [337]. The disease is self-limiting, and the mice that survive beyond day four show increased weight gain and are healthy by days 10-12 post-administration.

## **3.2. Genetically engineered models**

## *3.2.1. IL-10-/-*

In 1993, Kuhn et al. discovered that mice deficient in the anti-inflammatory cytokine IL-10 spontaneously develop enterocolitis [213]. This model has been popular in IBD research and this genetic deficiency has been crossed onto many different genetic backgrounds of mouse [339-345]. The availability of different strains has highlighted the role that genetics plays in enterocolitis, as the severity of disease is strain dependent. The order of severity from most severe to least severe is as follows: C3Bir > 129 > BALB/c or NOD/Lt > C57BL/ 6 or C57BL/10. In addition to strain differences, development of entercolitis is also de‐ pendent on the microbiota, as GF IL-10-/- do not develop enterocolitis and disease is atte‐ nuated after administration of antibiotics to IL-10-/- mice harboring a conventional microbiota [346, 347]. The lack of IL-10 does not affect the development of B or T cells, but its absence does result in a lack of regulatory T cells [213, 348]. Studies to under‐ stand the development of disease in these mice have shown that B cells (while present in high numbers in the lamina propria) are not needed for the initiation of disease, but that disease is mediated by CD4<sup>+</sup> T cells [342, 349]. Transfer studies using RAG2-/- mice as re‐ cipients have specifically shown that naive CD4<sup>+</sup> T cells are capable of inducing colitis, and CD45RBlow CD4+ T cells from IL-10-/- mice can induce disease in these RAG2-/- mice [348, 349]. This latter observation implicates IL-10 as a central mediator of regulatory T cells (CD25+ Foxp3+ CD4+ T cells). It has also been shown that IL-12 and IFN-γ are need‐ ed for initiation but not the continuation of colitis [342, 350]. The increase in the produc‐ tion of IL-12 and IFN-γ along with undetectable levels of IL-4 indicates that the enterocolitis in these mice is mediated by a Th1 immune response, similar to that ob‐ served in humans with IBD [342, 346].

large intestine. The mucosa, submucosa, and muscularis all presented with inflammatory cell infiltrates (macrophages and CD4+ T cells predominately), and there was also a loss of goblet cells, epithelial cell hyperplasia, and deep fissure ulcers. Elevated levels of IFNγ were present in these mice, and treatment with anti-IFN-γ or anti-TNF (α and β) anti‐ bodies ameliorated disease in these mice. However, the protection garnered by using the antibodies was not as great as when CD45RBlo CD4+ T cells were transferred along with CD45RBhi CD4+ T cells [362]. This disease type mimics CD with respect to the type of in‐ flammatory response generated (Th1) and the type of intestinal inflammation present (transmural infiltrate of CD4+ T cells) [365]. There is also a definite microbial component in this model of disease, as restricted microbiota mice have less severe disease as com‐

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Although not considered a microbial cause of IBD, the presence of *Helicobacter* spp. does adjuvant or predispose mice to the onset of colitis in some models. In our lab, we utilize a dual hit model of colitis consisting of both *Helicobacter bilis* colonization and low-dose (1.5%) DSS

**Figure 8.** To increase sensitivity to a colitic insult, ASF-bearing C3H/HeN:Tac mice were colonized with a bacterial pro‐

vocateur, *Helicobater bilis*. An otherwise non-colitic low dose of DSS is then administered resulting in colitis.

pared to their SPF counterparts [364].

to elicit colitis, as shown in Figure 8 [366].

**3.4. Bacterial-induced models**

*3.4.1. Helicobacter spp.*

## *3.2.2. Mdr1a-/-*

In 1994, mice lacking the gene *mdr1a* were generated. This gene encodes a P-glycoprotein, which is a drug efflux pump, thus protecting host cells from the build-up of toxic compounds. It was noted later that these mice spontaneously develop a colitis similar to human IBD and that disease can be exacerbated by colonization with *Helicobacter bilis* [351, 352]. These *mdr1a-/* mice have reduced growth rates compared to their WT counterparts and their histological lesions begin in the proximal colon and proceed distally as disease severity progresses. *Mdr1a-/* males are more susceptible to the onset of severe disease than females [353, 354]. *Mdr1a-/-* mice also have increased epithelial permeability and reduced phosphorylation of both occludin and ZO-1, tight junction proteins, compared to WT counterparts [354]. They exhibit increased bacterial translocation with bacteria detected in both the spleen and lymph nodes that correlates with disease severity. Therefore, the induction of disease is associated with a defect in the epithelial barrier function of the GI tract. In addition to the similar disease progression found in IBD patients, studying these mice are of interest because the human MDR1 gene has been mapped on a loci that that is associated with susceptibility of IBD, although this rela‐ tionship is under debate [355-358].

## *3.2.3. TRUC*

TRUC mice are both T-bet-/- and RAG-/- and spontaneously develop colitis by four weeks of age [359, 360]. T-bet (T-box expressed in T cells) is a transcription factor that aids in the development of a Th1 response [359]. These mice have increased permeability of their colonic epithelium that increases with age and increased rate of epithelial cell death. Microscopically, there is inflammatory cell infiltrate, goblet cell dropout, crypt loss, and the presences of ulcers. The only cytokine elevated in these mice is TNF-α and disease can be ameliorated using an anti-TNF-α antibody. The microbiota also contributes to disease in this model, as treatment with antibiotics was able to "cure" the mice of their colitis. Additionally, the TRUC colitic microbiota can be horizontally transferred to both WT and RAG-/- mice, and 16S rRNA analysis of feces from TRUC mice revealed that the presence of *Klebsiella pneumoniae* and *Proteus mirabilis* correlate with colitis. Interestingly, GF TRUC mice colonized with these two organ‐ isms alone do not develop colitis unless a more complete microbiota is present [361].

#### **3.3. CD45RBhi CD4+ T-cell transfer model**

C.B.-17 *scid* mice administered CD45RBhi CD4+ T cells develop a wasting disease which is not seen if CD45RBlo CD4+ T cells or CD45RBhi CD8+ T cells are adoptively transferred [362-364]. Disease occurs three to five weeks post-administration and is limited to the large intestine. The mucosa, submucosa, and muscularis all presented with inflammatory cell infiltrates (macrophages and CD4+ T cells predominately), and there was also a loss of goblet cells, epithelial cell hyperplasia, and deep fissure ulcers. Elevated levels of IFNγ were present in these mice, and treatment with anti-IFN-γ or anti-TNF (α and β) anti‐ bodies ameliorated disease in these mice. However, the protection garnered by using the antibodies was not as great as when CD45RBlo CD4+ T cells were transferred along with CD45RBhi CD4+ T cells [362]. This disease type mimics CD with respect to the type of in‐ flammatory response generated (Th1) and the type of intestinal inflammation present (transmural infiltrate of CD4+ T cells) [365]. There is also a definite microbial component in this model of disease, as restricted microbiota mice have less severe disease as com‐ pared to their SPF counterparts [364].

#### **3.4. Bacterial-induced models**

## *3.4.1. Helicobacter spp.*

[348, 349]. This latter observation implicates IL-10 as a central mediator of regulatory T cells (CD25+ Foxp3+ CD4+ T cells). It has also been shown that IL-12 and IFN-γ are need‐ ed for initiation but not the continuation of colitis [342, 350]. The increase in the produc‐ tion of IL-12 and IFN-γ along with undetectable levels of IL-4 indicates that the enterocolitis in these mice is mediated by a Th1 immune response, similar to that ob‐

In 1994, mice lacking the gene *mdr1a* were generated. This gene encodes a P-glycoprotein, which is a drug efflux pump, thus protecting host cells from the build-up of toxic compounds. It was noted later that these mice spontaneously develop a colitis similar to human IBD and that disease can be exacerbated by colonization with *Helicobacter bilis* [351, 352]. These *mdr1a-/* mice have reduced growth rates compared to their WT counterparts and their histological lesions begin in the proximal colon and proceed distally as disease severity progresses. *Mdr1a-/* males are more susceptible to the onset of severe disease than females [353, 354]. *Mdr1a-/-* mice also have increased epithelial permeability and reduced phosphorylation of both occludin and ZO-1, tight junction proteins, compared to WT counterparts [354]. They exhibit increased bacterial translocation with bacteria detected in both the spleen and lymph nodes that correlates with disease severity. Therefore, the induction of disease is associated with a defect in the epithelial barrier function of the GI tract. In addition to the similar disease progression found in IBD patients, studying these mice are of interest because the human MDR1 gene has been mapped on a loci that that is associated with susceptibility of IBD, although this rela‐

TRUC mice are both T-bet-/- and RAG-/- and spontaneously develop colitis by four weeks of age [359, 360]. T-bet (T-box expressed in T cells) is a transcription factor that aids in the development of a Th1 response [359]. These mice have increased permeability of their colonic epithelium that increases with age and increased rate of epithelial cell death. Microscopically, there is inflammatory cell infiltrate, goblet cell dropout, crypt loss, and the presences of ulcers. The only cytokine elevated in these mice is TNF-α and disease can be ameliorated using an anti-TNF-α antibody. The microbiota also contributes to disease in this model, as treatment with antibiotics was able to "cure" the mice of their colitis. Additionally, the TRUC colitic microbiota can be horizontally transferred to both WT and RAG-/- mice, and 16S rRNA analysis of feces from TRUC mice revealed that the presence of *Klebsiella pneumoniae* and *Proteus mirabilis* correlate with colitis. Interestingly, GF TRUC mice colonized with these two organ‐

isms alone do not develop colitis unless a more complete microbiota is present [361].

C.B.-17 *scid* mice administered CD45RBhi CD4+ T cells develop a wasting disease which is not seen if CD45RBlo CD4+ T cells or CD45RBhi CD8+ T cells are adoptively transferred [362-364]. Disease occurs three to five weeks post-administration and is limited to the

 **T-cell transfer model**

served in humans with IBD [342, 346].

tionship is under debate [355-358].

*3.2.2. Mdr1a-/-*

108 Inflammatory Bowel Disease

*3.2.3. TRUC*

**3.3. CD45RBhi CD4+**

Although not considered a microbial cause of IBD, the presence of *Helicobacter* spp. does adjuvant or predispose mice to the onset of colitis in some models. In our lab, we utilize a dual hit model of colitis consisting of both *Helicobacter bilis* colonization and low-dose (1.5%) DSS to elicit colitis, as shown in Figure 8 [366].

**Figure 8.** To increase sensitivity to a colitic insult, ASF-bearing C3H/HeN:Tac mice were colonized with a bacterial pro‐ vocateur, *Helicobater bilis*. An otherwise non-colitic low dose of DSS is then administered resulting in colitis.

mice compared to the uninfected control *mdr1a-/-* mice [352]. The disease phenotypes between spontaneous versus *Helicobacter*-induced colitis in *mdr1a-/-* mice were different, with epithelial ulceration not present in the *Helicobacter-*induced colitis. When *mdr1a-/-* mice were co-colonized with both *Helicobacter* species, the morbidity and mortality rate was between that of mice colonized by either *H. bilis* or *H. hepaticus* and the colitis that developed was characterized by dysplasia [372]. *H. bilis* was also able to out compete *H. hepaticus in vivo* as evidenced by recovery of higher numbers of *H. bilis*, suggesting that these two species may compete for

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Additionally, colonization of IL-10-/- C57BL/6 mice with *Helicobacter* species results in the induction of colitis in otherwise disease-free mice [373]. It was also shown that the onset and severity of colitis was species specific in relation to colonization by *H. bilis* or *H. hepaticus* [374]. With respect to C3Bir.129 IL-10-/- mice, the presence of *Helicobacter* spp. is required for the spontaneous onset of disease [339]. Further studies show that colonization of RAG1-/- mice with *Helicobacter* species fails to elicit clinical signs of disease after nine months of colonization and only very mild colonic inflammation was detected at necropsy [374]. Taken together, these observations indicate that disease induction can be mediated by an aberrant adaptive immune response initiated by bacterial provocateurs entering an otherwise stable host-microbe

Similar to the need for the a resident microbiota in the TRUC model of colitis, mice colonized with *Brachyspira hyodysenteriae* also require the presence of a microbiota for the induction of typhlocolitis. *B. hyodysenteriae* is an anaerobic spirochete that is the causative agent of swine dysentery [375]. While the pathogenesis of *B. hyodysenteriae* is associated with the production of a ß-hemolysin [376], disease does not develop in the absence of a resident microbiota as was demonstrated by the inoculation of germfree pigs [377-379]. The importance of the microbiota was further demonstrated when the microbiota of C3H/HeSnJ or BALB/c mice was depleted by adding a cocktail of antibiotics (rifampicin, colistin, spectinomycin, spiramycin, and vancomycin) to their drinking water. As can be seen in Figure 10, the antibiotic cocktail depleted the numbers of Gram-positive, Gram-negative, and strict anaerobes by as much as 5 to 7 log10 (Nibbelink and Wannemuehler, unpublished observations). On day seven, antibiotic treated and sham treated mice were inoculated with 1 x 108 *B. hyodysenteirae* strain B204 and severity of disease was evaluated at 5, 10, and 15 days post-infection (DPI). As can be seen in Figure 11, the sham-treated mice developed severe typhlocolitis while the antibiotic treated mice had no lesions. At 15 DPI, the antibiotics were withdrawn from the drinking water of the remaining *B. hyodysenteriae-*infected mice to allow the microbiota to recover. On day 30 PI, the mice that had been treated with antibiotics through 15 DPI now presented with severe typhlocolitis. The presence and severity of typhlocolitis in these mice correlated with the presence of TNF-specific mRNA in the cecal tissue of the *B. hyodysenteriae-*infected mice (data not shown). Lastly, *B. hyodysenteriae-*induced typhlocolitis could also be prevented when the host's inflammatory response was inhibited [380, 381]. Collectively, these observations indicate that certain bacterial provocateurs (*K. pneumoniae, P. mirabilis,* and *B. hyodysenteriae)*

similar niches in the GIT.

environment.

*3.4.2. Brachyspira hyodysenteriae*

**Figure 9.** Mice colonized with *Helicobacter bilis* and subsequently administered a low dose of DSS exhibit colitis that is not seen with *H. bilis* or DSS alone. Disease is characterized grossly by colonic shortening, edema, cecal atrophy, en‐ larged lymphoid aggregates, and blood present in the contents. Microscopically, the disease presents with inflamma‐ tory cell infiltrate, crypt hyperplasia, glandular dropout, and cellular erosion

It has been noted that colonization of mice with *H. bilis* alone can change mucosal gene expression and alter the immune response in C3H/HeN mice bearing the altered Schaedler's flora (ASF) [366, 369]. Genes involved in T cell receptor signaling, the survival and activation of peripheral B cells, and chemotaxis are a few examples of the host genes that were upregu‐ lated by *H. bilis* colonization. Genes that were down regulated were involved in fatty acid metabolism and detoxification. After *H. bilis* colonization, serum antibodies directed at antigens derived from members of the ASF are induced [366, 369]. *H. hepaticus* colonization of A/JCr mice also induced changes in gene expression in the cecum, with female mice being more susceptible to the onset of disease [370, 371].

It appears that an over zealous host response to the introduction of the novel organism (i.e., provocateur) predisposes certain strains of mice for the onset of typholocoltiis following a secondary colitic insult. In a study comparing A/JCr mice (that develop mild inflammation) to C57BL/6 (who do not develop inflammation) mice, cecal gene expression profiles revealed that A/JCr mice had more genes differentially regulated (176) compared to C57BL/6 (80). Differ‐ entially expressed genes were predominantly those associated with immune response, chemotaxis, signal transduction, and antigen processing in the A/JCr mice while the genes upregulated in the C57BL/6 mice were predominately associated with immunoglobulin production.

In the *mdr1a-/-* mouse, it is not simply the presence of *Helicobacter* that results in the induction of colitis but the specific species of *Helicobacter* influences the induction of a differential immune response. In *mdr1a-/-* mice, *H. bilis* colonization causes colitis as early as three weeks post-infection, whereas *H. hepaticus* colonization ameliorated the severity of colitis in these mice compared to the uninfected control *mdr1a-/-* mice [352]. The disease phenotypes between spontaneous versus *Helicobacter*-induced colitis in *mdr1a-/-* mice were different, with epithelial ulceration not present in the *Helicobacter-*induced colitis. When *mdr1a-/-* mice were co-colonized with both *Helicobacter* species, the morbidity and mortality rate was between that of mice colonized by either *H. bilis* or *H. hepaticus* and the colitis that developed was characterized by dysplasia [372]. *H. bilis* was also able to out compete *H. hepaticus in vivo* as evidenced by recovery of higher numbers of *H. bilis*, suggesting that these two species may compete for similar niches in the GIT.

Additionally, colonization of IL-10-/- C57BL/6 mice with *Helicobacter* species results in the induction of colitis in otherwise disease-free mice [373]. It was also shown that the onset and severity of colitis was species specific in relation to colonization by *H. bilis* or *H. hepaticus* [374]. With respect to C3Bir.129 IL-10-/- mice, the presence of *Helicobacter* spp. is required for the spontaneous onset of disease [339]. Further studies show that colonization of RAG1-/- mice with *Helicobacter* species fails to elicit clinical signs of disease after nine months of colonization and only very mild colonic inflammation was detected at necropsy [374]. Taken together, these observations indicate that disease induction can be mediated by an aberrant adaptive immune response initiated by bacterial provocateurs entering an otherwise stable host-microbe environment.

## *3.4.2. Brachyspira hyodysenteriae*

**Figure 9.** Mice colonized with *Helicobacter bilis* and subsequently administered a low dose of DSS exhibit colitis that is not seen with *H. bilis* or DSS alone. Disease is characterized grossly by colonic shortening, edema, cecal atrophy, en‐ larged lymphoid aggregates, and blood present in the contents. Microscopically, the disease presents with inflamma‐

It has been noted that colonization of mice with *H. bilis* alone can change mucosal gene expression and alter the immune response in C3H/HeN mice bearing the altered Schaedler's flora (ASF) [366, 369]. Genes involved in T cell receptor signaling, the survival and activation of peripheral B cells, and chemotaxis are a few examples of the host genes that were upregu‐ lated by *H. bilis* colonization. Genes that were down regulated were involved in fatty acid metabolism and detoxification. After *H. bilis* colonization, serum antibodies directed at antigens derived from members of the ASF are induced [366, 369]. *H. hepaticus* colonization of A/JCr mice also induced changes in gene expression in the cecum, with female mice being

It appears that an over zealous host response to the introduction of the novel organism (i.e., provocateur) predisposes certain strains of mice for the onset of typholocoltiis following a secondary colitic insult. In a study comparing A/JCr mice (that develop mild inflammation) to C57BL/6 (who do not develop inflammation) mice, cecal gene expression profiles revealed that A/JCr mice had more genes differentially regulated (176) compared to C57BL/6 (80). Differ‐ entially expressed genes were predominantly those associated with immune response, chemotaxis, signal transduction, and antigen processing in the A/JCr mice while the genes upregulated in the C57BL/6 mice were predominately associated with immunoglobulin

In the *mdr1a-/-* mouse, it is not simply the presence of *Helicobacter* that results in the induction of colitis but the specific species of *Helicobacter* influences the induction of a differential immune response. In *mdr1a-/-* mice, *H. bilis* colonization causes colitis as early as three weeks post-infection, whereas *H. hepaticus* colonization ameliorated the severity of colitis in these

tory cell infiltrate, crypt hyperplasia, glandular dropout, and cellular erosion

more susceptible to the onset of disease [370, 371].

production.

110 Inflammatory Bowel Disease

Similar to the need for the a resident microbiota in the TRUC model of colitis, mice colonized with *Brachyspira hyodysenteriae* also require the presence of a microbiota for the induction of typhlocolitis. *B. hyodysenteriae* is an anaerobic spirochete that is the causative agent of swine dysentery [375]. While the pathogenesis of *B. hyodysenteriae* is associated with the production of a ß-hemolysin [376], disease does not develop in the absence of a resident microbiota as was demonstrated by the inoculation of germfree pigs [377-379]. The importance of the microbiota was further demonstrated when the microbiota of C3H/HeSnJ or BALB/c mice was depleted by adding a cocktail of antibiotics (rifampicin, colistin, spectinomycin, spiramycin, and vancomycin) to their drinking water. As can be seen in Figure 10, the antibiotic cocktail depleted the numbers of Gram-positive, Gram-negative, and strict anaerobes by as much as 5 to 7 log10 (Nibbelink and Wannemuehler, unpublished observations). On day seven, antibiotic treated and sham treated mice were inoculated with 1 x 108 *B. hyodysenteirae* strain B204 and severity of disease was evaluated at 5, 10, and 15 days post-infection (DPI). As can be seen in Figure 11, the sham-treated mice developed severe typhlocolitis while the antibiotic treated mice had no lesions. At 15 DPI, the antibiotics were withdrawn from the drinking water of the remaining *B. hyodysenteriae-*infected mice to allow the microbiota to recover. On day 30 PI, the mice that had been treated with antibiotics through 15 DPI now presented with severe typhlocolitis. The presence and severity of typhlocolitis in these mice correlated with the presence of TNF-specific mRNA in the cecal tissue of the *B. hyodysenteriae-*infected mice (data not shown). Lastly, *B. hyodysenteriae-*induced typhlocolitis could also be prevented when the host's inflammatory response was inhibited [380, 381]. Collectively, these observations indicate that certain bacterial provocateurs (*K. pneumoniae, P. mirabilis,* and *B. hyodysenteriae)*

may fail to induce disease in the absence of an appropriate resident microbiota. As depicted in Figure 12, the etiology of colitis is complex and may require the presence of a microbial provocateur, the resident microbiota, and a host inflammatory response.

Laboratories [54]. This lack of consistency in microbiota from mice of the same strain has even been identified at different facilities from the same vendor (Overstreet and Wannemuehler unpublished observation). Therefore, comparison of studies evaluating the microbiota of mice are difficult because there are hundreds of unknown bacterial species present in the murine microbiota and there is no standardized microbiota used by investigators. The advent of nextgen sequencing has helped to alleviate some of this challenge as all of the organisms in the gut can be identified based on 16S rRNA sequences. However, it still does not resolve the issue related to the use of disparate strains of mice with varying microbiota from different suppliers.

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**Figure 11.** Assessment of *Brachyspira hyodysenteriae-*induced typhlocolitis in C3H/HeSnJ mice treated with an antibi‐ otic cocktail. While *B. hyodysenteriae* colonized the antibiotic-treated mice to the same extent as it did the sham-treat‐ ed mice, there was no evidence of typhlocolitis in the antibiotic treated mice through 15 days post-infection (DPI). However, 15 days after the antibiotics were withdrawn (30 DPI), the mice that had been treated with the antibiotics

had developed severe disease once the microbiota recovered.

**Figure 10.** To assess the role of the microbiota in *Brachyspira hyodysenteriae-*induced typhlocolitis, C3H/HeSnJ mice were treated with an antibiotic cocktail to deplete the resident microbiota. Six days later, mice were infected with *B. hyodysenteriae.* The data indicate that there was a five to seven log10 reduction in the resident microbiota.

## **3.5. Conventional mice**

The majority of commercially available mice harbor a "conventional" microbiota. This simply means that the composition of the community is unknown. There are different types of conventionally-reared mice. For example, Taconic Farms maintain two types of conventional microbiota mice, restricted flora™ (RF) and murine pathogen free™ (PF). RF mice are not colonized by β-hemolytic *Streptococcus* species, *K. pneumoniae, K. oxytoca, Pseudomonas aerugi‐ nosa,* or *Staphylococcus aureus.* Mice that are PF are *Helicobacter* free but can contain organisms not found in RF mice [382]. Additionally, mice on the same background purchased from different vendors can harbor different microbiota, as highlighted in work by Ivanov and colleagues demonstrating the presence of SFB in C57BL/6 mice from Taconic but not Jackson Laboratories [54]. This lack of consistency in microbiota from mice of the same strain has even been identified at different facilities from the same vendor (Overstreet and Wannemuehler unpublished observation). Therefore, comparison of studies evaluating the microbiota of mice are difficult because there are hundreds of unknown bacterial species present in the murine microbiota and there is no standardized microbiota used by investigators. The advent of nextgen sequencing has helped to alleviate some of this challenge as all of the organisms in the gut can be identified based on 16S rRNA sequences. However, it still does not resolve the issue related to the use of disparate strains of mice with varying microbiota from different suppliers.

may fail to induce disease in the absence of an appropriate resident microbiota. As depicted in Figure 12, the etiology of colitis is complex and may require the presence of a microbial

**Figure 10.** To assess the role of the microbiota in *Brachyspira hyodysenteriae-*induced typhlocolitis, C3H/HeSnJ mice were treated with an antibiotic cocktail to deplete the resident microbiota. Six days later, mice were infected with *B.*

The majority of commercially available mice harbor a "conventional" microbiota. This simply means that the composition of the community is unknown. There are different types of conventionally-reared mice. For example, Taconic Farms maintain two types of conventional microbiota mice, restricted flora™ (RF) and murine pathogen free™ (PF). RF mice are not colonized by β-hemolytic *Streptococcus* species, *K. pneumoniae, K. oxytoca, Pseudomonas aerugi‐ nosa,* or *Staphylococcus aureus.* Mice that are PF are *Helicobacter* free but can contain organisms not found in RF mice [382]. Additionally, mice on the same background purchased from different vendors can harbor different microbiota, as highlighted in work by Ivanov and colleagues demonstrating the presence of SFB in C57BL/6 mice from Taconic but not Jackson

*hyodysenteriae.* The data indicate that there was a five to seven log10 reduction in the resident microbiota.

**3.5. Conventional mice**

112 Inflammatory Bowel Disease

provocateur, the resident microbiota, and a host inflammatory response.

**Figure 11.** Assessment of *Brachyspira hyodysenteriae-*induced typhlocolitis in C3H/HeSnJ mice treated with an antibi‐ otic cocktail. While *B. hyodysenteriae* colonized the antibiotic-treated mice to the same extent as it did the sham-treat‐ ed mice, there was no evidence of typhlocolitis in the antibiotic treated mice through 15 days post-infection (DPI). However, 15 days after the antibiotics were withdrawn (30 DPI), the mice that had been treated with the antibiotics had developed severe disease once the microbiota recovered.

Although the information gathered from these studies has been useful in analyzing the immune response to specific organisms, trying to relate the resultant disease to that char‐ acteristic of IBD is marginal at best because of the differences in the complexities of the microbiota. IBD itself is a multi-factorial disease and it has been fairly well-established that the role of bacteria in IBD is associated with a shift in community dynamics (i.e., dysbiosis) and not the presence/absence of one particular species. A perfect example of this complexity is the fact that *K. pneumonia* and *P. mirabilis* fail to induce colitis in GF

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Also referred to as gnotobiotic, defined microbiota (DM) mice have a microbiota in which all members are known and are housed in flexible film isolators to maintain this status [388]. One of the most established DM mouse models harbors the "Altered Schaedler Flora" (ASF). Developed by Dr. Rodger Orcutt and colleagues as a request from the National Cancer Institute, these eight microbial species were originally used to standardized the microbiota of the rodents used as founders in their breeding colonies [389]. He chose to modify his mentor's (Dr. Russ Schaedler) cocktail of organisms by eliminating facultative anaerobes (*Escherichia coli* var. mutabilis and *Streptococcus fecalis*) and the anaerobic *Streptococcus* and *Clostridia* spp. that formed the original "Schaedler Flora". Dr. Orcutt added four additional species (see Table 4 below). This resulted in an anaerobic community devoid of any cocci or spore-forming blunt ended rods, which comprise the main isolator contaminants, making it easier to monitor for contamination of the gnotobiotic isolator. This new community was dubbed the "Altered Schaedler Flora" and was utilized in the breeding stock by major commercial mouse vendors in the US in that era. The ASF consists of members with different morphologies so that identification using fecal smears for microscopic evaluation was possible. The members, whose full genomes have yet to be sequenced, have had 16S rRNA sequencing performed to assist

Multiple studies have demonstrated the stability of this community both when maintained under gnotobiotic housing conditions or when part of a conventionalized microbiota [391-394]. In our own lab, all eight ASF members have been stably maintained in our breeding colony for over 12 years, indicating the remarkable stability of this model microbial community over time. PCR primers have been developed for each of the eight ASF members as well as groupspecific FISH probes [391, 394]. Therefore, the effects of any perturbation of the ASF, such as with antibiotics, inflammation, or CAM treatments can be monitored by bacterial abundance as well as spatial redistribution using qPCR and FISH, respectively. All of the organisms can be cultured and whole cell sonicates produced to measure the immune response to each organism individually (something that is impossible to do with a conventional microbiota). Additionally, this community, although limited in scope, is able to synthesize all the metabo‐ lites needed by the mouse and maintains near normal cecal shape and size, something not possible in GF mice. It is important to note, however, that some of the characteristics of ASF mice are more similar to GF than conventional mice. Both ASF and GF mice have high fecal tryptic activity and possess the ability to degrade mucin and β-aspartylglycine. They also

TRUC mice (Figure 12) [361].

**3.7. Defined microbiota mice**

with the identification of the organisms [390].

**Figure 12.** The pathogenesis of inflammatory bowel disease is complex. Studies from animal models indicate that the etiology of disease involves the presence of a bacterial provocateur, the resident microbiota, and the host response.

#### **3.6. Germfree mice**

GF mice are completely devoid of microbial life. As mentioned previously, this does grossly affect the anatomy of these mice, which is most evident by the enlarged cecum being the most prominent feature [34]. The discovery that many GF mouse strains that carry genetic deficiencies associated with IBD (notably the IL-10-/-) do not develop colitis has led to the popularity of GF models to study the role of bacteria in the pathogenesis of IBD [346]. To determine if an organism is capable of initiating colitis, GF mice are monoassociated with a single bacterial species and then monitored for clinical signs of disease. Some of the bacterial strains used to date to evaluate their ability to induce dis‐ ease in IL-10-/- mice are shown in Table 3.

**Table 3.** Bacterial strains used to monoassociate GF mice to examine the ability of the strain to induce colitis

Although the information gathered from these studies has been useful in analyzing the immune response to specific organisms, trying to relate the resultant disease to that char‐ acteristic of IBD is marginal at best because of the differences in the complexities of the microbiota. IBD itself is a multi-factorial disease and it has been fairly well-established that the role of bacteria in IBD is associated with a shift in community dynamics (i.e., dysbiosis) and not the presence/absence of one particular species. A perfect example of this complexity is the fact that *K. pneumonia* and *P. mirabilis* fail to induce colitis in GF TRUC mice (Figure 12) [361].

## **3.7. Defined microbiota mice**

**Figure 12.** The pathogenesis of inflammatory bowel disease is complex. Studies from animal models indicate that the etiology of disease involves the presence of a bacterial provocateur, the resident microbiota, and the host response.

GF mice are completely devoid of microbial life. As mentioned previously, this does grossly affect the anatomy of these mice, which is most evident by the enlarged cecum being the most prominent feature [34]. The discovery that many GF mouse strains that carry genetic deficiencies associated with IBD (notably the IL-10-/-) do not develop colitis has led to the popularity of GF models to study the role of bacteria in the pathogenesis of IBD [346]. To determine if an organism is capable of initiating colitis, GF mice are monoassociated with a single bacterial species and then monitored for clinical signs of disease. Some of the bacterial strains used to date to evaluate their ability to induce dis‐

> **Organism Colitis Severity Time of Disease Onset Reference** *Enterococcus faecalis* Severe 10-12 weeks p.i. [383, 384]

*Escherichia coli (*from WT SPF mouse) Moderate 3 weeks p.i. [383] *Pseudomonas flourescens* No disease - [383] *Helicobacter hepaticus* No disease - [385] *Lactococcus lactis* No disease - [384] *Viridans* group *Streptococcus* No disease - [386] *Clostridium sordellii* No disease - [386] *Bacteroides vulgatus* No disease - [386] *Bifidobacterium animalis* Mild 23 weeks p.i. [387] *Bifidobacterium infantis* No disease - [387]

**Table 3.** Bacterial strains used to monoassociate GF mice to examine the ability of the strain to induce colitis

**3.6. Germfree mice**

114 Inflammatory Bowel Disease

ease in IL-10-/- mice are shown in Table 3.

Also referred to as gnotobiotic, defined microbiota (DM) mice have a microbiota in which all members are known and are housed in flexible film isolators to maintain this status [388]. One of the most established DM mouse models harbors the "Altered Schaedler Flora" (ASF). Developed by Dr. Rodger Orcutt and colleagues as a request from the National Cancer Institute, these eight microbial species were originally used to standardized the microbiota of the rodents used as founders in their breeding colonies [389]. He chose to modify his mentor's (Dr. Russ Schaedler) cocktail of organisms by eliminating facultative anaerobes (*Escherichia coli* var. mutabilis and *Streptococcus fecalis*) and the anaerobic *Streptococcus* and *Clostridia* spp. that formed the original "Schaedler Flora". Dr. Orcutt added four additional species (see Table 4 below). This resulted in an anaerobic community devoid of any cocci or spore-forming blunt ended rods, which comprise the main isolator contaminants, making it easier to monitor for contamination of the gnotobiotic isolator. This new community was dubbed the "Altered Schaedler Flora" and was utilized in the breeding stock by major commercial mouse vendors in the US in that era. The ASF consists of members with different morphologies so that identification using fecal smears for microscopic evaluation was possible. The members, whose full genomes have yet to be sequenced, have had 16S rRNA sequencing performed to assist with the identification of the organisms [390].

Multiple studies have demonstrated the stability of this community both when maintained under gnotobiotic housing conditions or when part of a conventionalized microbiota [391-394]. In our own lab, all eight ASF members have been stably maintained in our breeding colony for over 12 years, indicating the remarkable stability of this model microbial community over time. PCR primers have been developed for each of the eight ASF members as well as groupspecific FISH probes [391, 394]. Therefore, the effects of any perturbation of the ASF, such as with antibiotics, inflammation, or CAM treatments can be monitored by bacterial abundance as well as spatial redistribution using qPCR and FISH, respectively. All of the organisms can be cultured and whole cell sonicates produced to measure the immune response to each organism individually (something that is impossible to do with a conventional microbiota). Additionally, this community, although limited in scope, is able to synthesize all the metabo‐ lites needed by the mouse and maintains near normal cecal shape and size, something not possible in GF mice. It is important to note, however, that some of the characteristics of ASF mice are more similar to GF than conventional mice. Both ASF and GF mice have high fecal tryptic activity and possess the ability to degrade mucin and β-aspartylglycine. They also cannot convert bilirubin to urobilinogens or cholesterol to coprostanol [395]. Interestingly, this same research team compared these parameters in CD patients versus healthy subjects and the characteristics of the microbial metabolism associated with the microbiota of CD patients were very similar to those of the ASF [396]. This study suggests that there is benefit to the use of the ASF in mouse models of IBD.

munity. This addition caused the reduction of the original *E. coli* to drop to undetectable levels at day two post-addition. Later *L. johnsonii* also reached undetectable levels. *B. longum* decreased initially but them returned to normal titer levels. Lastly, when the original triassociated mice were exposed to conventional mouse feces, the numbers of the original three

In this chapter, we have discussed the three most common types of microbiota available for use in IBD research. Because it is clear that IBD is associated with an imbalance in the microbial community, the use of GF mice (which cannot mimic a "community" dynamic) may be less useful in unraveling the complexities of a multifactorial disease in place of defined microbiota and conventional microbiota mice. Ideally, one would want to use a simplified community (such as the ASF) where the actions of all organisms can be assessed. To understand the dynamic interactions that occur between microbes and between the microbes and the host, it will be important to start with what is known in order to begin the unraveling the enigmatic

, Amanda E. Ramer-Tait3

1 Department of Veterinary Microbiology and Preventive Medicine, Iowa State University,

3 Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln,

[1] Whitman, W.B., Coleman, D.C., and Wiebe, W.J., Prokaryotes: the unseen majority. Proceedings of the National Academy of Sciences of the United States of America,

[2] Savage, D.C., Microbial ecology of the gastrointestinal tract. Annual Review of Mi‐

[3] Zhu, B., Wang, X., and Li, L., Human gut microbiome: the second genome of human

[4] Structure, function and diversity of the healthy human microbiome. Nature, 2012.

2 Department of Veterinary Clinical Sciences, Iowa State University, Ames, IA, USA

, Albert E. Jergens2

and

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organisms decreased.

**Author details**

Ames, IA, USA

NE, USA

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