Gastrointestinal release site for delayed release and gelatin capsules in healthy dogs.

Gelatin capsules deliver their contents to the stomach, while delayed-release (DR) capsules are designed to allow delivery to the small intestine. This study evaluated the gastrointestinal release site of DR capsules in six healthy adult dogs compared to gelatin capsules. Both gelatin and DR capsules were filled with barium-impregnated polyethylene spheres (BIPS™), and following enteral administration, release site was assessed using abdominal radiographs at baseline, immediately after ingestion, 15 min post-ingestion, 30 min post-ingestion, and then every 30 min thereafter. The evaluated phases included fasted conditions (phase 1, n = 6), increased meal size (phase 2, n = 2), double encapsulation (phase 3, n = 2), and altered capsule size (phase 4, n = 1). The released site was the stomach in all phases for both capsule types. In phase 1, DR capsules had a significantly prolonged time (median 60 min, range 60-90) to release BIPS™ compared to gelatin capsules (15 min, range 15-30; p = .03). In phase 2 (full meal size), 3 (double encapsulation), and 4 (smaller capsule size) pilot studies, release time was prolonged but still occurred in the stomach. This is similar to the release site for gelatin capsules but differs from the release site for DR capsules in people. This has implications for pharmacologic outcomes for products that are affected by gastric physiology (e.g. fecal microbiota transplantation). Based on this pilot data, clinicians and researchers should not assume DR capsules will allow for intestinal delivery of contents in dogs. Future studies should be conducted on larger and varied populations of dogs.

Untargeted metabolomic profiling of serum from client-owned cats with early and late-stage chronic kidney disease.

Evaluation of the metabolome could discover novel biomarkers of disease. To date, characterization of the serum metabolome of client-owned cats with chronic kidney disease (CKD), which shares numerous pathophysiological similarities to human CKD, has not been reported. CKD is a leading cause of feline morbidity and mortality, which can be lessened with early detection and appropriate treatment. Consequently, there is an urgent need for early-CKD biomarkers. The goal of this cross-sectional, prospective study was to characterize the global, non-targeted serum metabolome of cats with early versus late-stage CKD compared to healthy cats. Analysis revealed distinct separation of the serum metabolome between healthy cats, early-stage and late-stage CKD. Differentially abundant lipid and amino acid metabolites were the primary contributors to these differences and included metabolites central to the metabolism of fatty acids, essential amino acids and uremic toxins. Correlation of multiple lipid and amino acid metabolites with clinical metadata important to CKD monitoring and patient treatment (e.g. creatinine, muscle condition score) further illustrates the relevance of exploring these metabolite classes further for their capacity to serve as biomarkers of early CKD detection in both feline and human populations.

Collaborative Metabolism: Gut Microbes Play a Key Role in Canine and Feline Bile Acid Metabolism.

Bile acids, produced by the liver and secreted into the gastrointestinal tract, are dynamic molecules capable of impacting the overall health of dogs and cats in many contexts. Importantly, the gut microbiota metabolizes host primary bile acids into chemically distinct secondary bile acids. This review explores the emergence of new literature connecting microbial-derived bile acid metabolism to canine and feline health and disease. Moreover, this review highlights multi-omic methodologies for translational research as an area for continued growth in veterinary medicine aimed at accelerating microbiome science and medicine as it pertains to bile acid metabolism in dogs and cats.

Clinician prescribing practices for managing canine idiopathic acute diarrhea are not evidence based.

Objective: To characterize clinician preferences and justification for preferred methods for managing canine idiopathic acute diarrhea (IAD) and compare results to evidence-based literature. sample: 284 surveys from veterinarians in small animal first-opinion practice. Methods: Veterinarians were asked to complete a survey (61 questions) including background demographic information, practice type and location, duration in practice, and management questions for canine IAD pertaining to nutritional, probiotic, antimicrobial, antidiarrheal, benign neglect, and other therapies. The survey was available between May 5, 2021, and August 30, 2021. Results: Respondents reported that their preferred first-line therapy for canine IAD included dietary modification (41.3% of respondents), probiotics (20.1%), antimicrobials (21.2%), antidiarrheal medications (13.0%), and benign neglect (4.3%). The percentage of respondents who reported each therapy as either extremely effective or very effective for canine IAD varied by treatment, as follows: antimicrobials (75.2%), dietary modification (59.13%), antidiarrheal medications (42.5%), probiotics (35.5%), and benign neglect (6.52%). Perceptions of effectiveness, efficiency of treatment, and clinician justification for use were variable among treatments. Reported practice styles were occasionally in disagreement with evidence-based methods of canine IAD management. Clinical Relevance: Current clinical management of IAD is not consistently in agreement with evidence-based recommendations. The results of this study underscore the continued need to evaluate veterinary prescribing practice trends compared to evidence-based recommendations and promote dissemination of new information.

Lack of SARS-CoV-2 Viral RNA Detection among a Convenience Sampling of Ohio Wildlife, Companion, and Agricultural Animals, 2020-2021.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in humans in late 2019 and spread rapidly, becoming a global pandemic. A zoonotic spillover event from animal to human was identified as the presumed origin. Subsequently, reports began emerging regarding spillback events resulting in SARS-CoV-2 infections in multiple animal species. These events highlighted critical links between animal and human health while also raising concerns about the development of new reservoir hosts and potential viral mutations that could alter the virulence and transmission or evade immune responses. Characterizing susceptibility, prevalence, and transmission between animal species became a priority to help protect animal and human health. In this study, we coalesced a large team of investigators and community partners to surveil for SARS-CoV-2 in domestic and free-ranging animals around Ohio between May 2020 and August 2021. We focused on species with known or predicted susceptibility to SARS-CoV-2 infection, highly congregated or medically compromised animals (e.g., shelters, barns, veterinary hospitals), and animals that had frequent contact with humans (e.g., pets, agricultural animals, zoo animals, or animals in wildlife hospitals). This included free-ranging deer (n = 76 individuals), free-ranging mink (n = 57), multiple species of bats (n = 59), and other wildlife in addition to domestic cats (n = 275) and pigs (n = 184). In total, we tested 792 individual animals (34 species) via rRT-PCR for SARS-CoV-2 RNA. SARS-CoV-2 viral RNA was not detected in any of the tested animals despite a major peak in human SARS-CoV-2 cases that occurred in Ohio subsequent to the peak of animal samplings. Importantly, we did not test for SARS-CoV-2 antibodies in this study, which limited our ability to assess exposure. While the results of this study were negative, the surveillance effort was critical and remains key to understanding, predicting, and preventing the re-emergence of SARS-CoV-2 in humans or animals.

Measuring 25-hydroxyvitamin D in cats: comparison of a whole-blood lateral flow assay, 2 dried-blood-spot tests, and serum LC-MS/MS.

Measuring 25-hydroxyvitamin D (25D) can be a challenge in veterinary medicine because of laboratory accessibility and required sample volume. We compared 2 dried-blood-spot (DBS) tests and a lateral flow assay (LFA) to the gold standard, liquid chromatography-tandem mass spectrometry (LC-MS/MS). We hypothesized that there would be good agreement among the tests, within a clinically significant limit of agreement of ± 25 nmol/L. We collected blood from 6 healthy purpose-bred 2-y-old cats at 6 times over 6 wk, and measured 25D concentrations with all 4 tests. Agreement of the 3 candidate tests and LC-MS/MS was evaluated via Bland-Altman analysis, Passing-Bablok regression, and Lin correlation coefficients. Bland-Altman analysis demonstrated that the mean bias was >± 25 nmol/L for all 3 candidate tests in comparison to serum LC-MS/MS concentrations. The 95% CIs for the mean bias did not include zero, further supporting the presence of significant bias among methods. Additionally, all 3 tests had poor agreement with serum LC-MS/MS concentrations when analyzed by Lin correlation coefficient analysis, and bias between methods was further characterized by Passing-Bablok analysis. Based on these results, none of these 3 tests is recommended as an alternative to LC-MS/MS testing for 25D measurement in cats.

Fecal identification markers impact the feline fecal microbiota.

Fecal diagnostics are a mainstay of feline medicine, and fecal identification markers help to distinguish individuals in a multi-cat environment. However, the impact of identification markers on the fecal microbiota are unknown. Given the increased interest in using microbiota endpoints to inform diagnosis and treatment, the objective of this study was to examine the effects of orally supplemented glitter and crayon shavings on the feline fecal microbiota (amplicon sequencing of 16S rRNA gene V4 region). Fecal samples were collected daily from six adult cats that were randomized to receive oral supplementation with either glitter or crayon for two weeks, with a two-week washout before receiving the second marker. No adverse effects in response to marker supplementation were seen for any cat, and both markers were readily identifiable in the feces. Microbiota analysis revealed idiosyncratic responses to fecal markers, where changes in community structure in response to glitter or crayon could not be readily discerned. Given these findings, it is not recommended to administered glitter or crayon shavings as a fecal marker when microbiome endpoints are used, however their clinical use with other diagnostics should still be considered.

Dietary fiber aids in the management of canine and feline gastrointestinal disease.

Dietary fiber describes a diverse assortment of nondigestible carbohydrates that play a vital role in the health of animals and maintenance of gastrointestinal tract homeostasis. The main roles dietary fiber play in the gastrointestinal tract include physically altering the digesta, modulating appetite and satiety, regulating digestion, and acting as a microbial energy source through fermentation. These functions can have widespread systemic effects. Fiber is a vital component of nearly all commercial canine and feline diets. Key features of fiber types, such as fermentability, solubility, and viscosity, have been shown to have clinical implications as well as health benefits in dogs and cats. Practitioners should know how to evaluate a diet for fiber content and the current knowledge on fiber supplementation as it relates to common enteropathies including acute diarrhea, chronic diarrhea, constipation, and hairball management. Understanding the fundamentals of dietary fiber allows the practicing clinician to use fiber optimally as a management modality.

Home-cooked diets cost more than commercially prepared dry kibble diets for dogs with chronic enteropathies.

OBJECTIVE: Nutrition plays a fundamental role in the management of canine chronic enteropathies (CCEs). Dog owners may elect to feed home-cooked diets (HCDs) rather than veterinary commercially prepared diets (CPDs) because of perceived lower costs. There is a paucity of data comparing costs of these options. We hypothesize there will be differences in costs between complete and balanced HCDs and nutritionally comparable CPDs. SAMPLE: 6 Home-cooked diets. PROCEDURES: Six HCD recipes (2 highly digestible, 2 limited antigen, 2 low-fat) were formulated by 2 board-certified veterinary nutritionists to mimic the nutritional and ingredient profiles of veterinary CPDs for management of CCEs. The cost (in US$ on a per 100 kilocalorie [kcal] basis) of each recipe was determined via collection of ingredient prices from 3 grocery stores combined with supplement prices from online retailers. Prices of CPDs were obtained from a national online retailer. Maintenance energy requirements of 1.6 X (70 X BWkg0.75), where BWkg represents body weight in kilograms, were calculated for 3 dog sizes (5, 20, and 40 kg), and costs of feeding maintenance energy requirements with HCDs versus dry and canned CPDs were compared with a Kruskal-Wallis test and post hoc testing. RESULTS: The median costs of all dry and canned CPDs and HCDs were $0.29 (range, $0.18 to $0.46), $1.01 (range, $0.77 to $1.20), and $0.55 (range, $0.35 to $1.14), respectively. Feeding complete and balanced HCDs cost more than feeding dry CPDs (P < .001), but not canned CPDs (P > .99). CLINICAL RELEVANCE: Dry CPDs cost the least for nutritional management of CCEs. There is a wide range of costs for both CPDs and HCDs.

Escherichia coli probiotic exhibits in vitro growth-limiting effects on clinical feline uropathogenic E coli isolates.

OBJECTIVE: To characterize uropathogenic Escherichia coli (UPEC) in cases of clinical feline urinary tract infection (UTI) and subclinical bacteriuria and investigate the in vitro effects of E coli strain Nissle 1917 on isolate growth. ANIMALS: 40 cats with positive E coli culture results for urine collected during routine evaluation. PROCEDURES: Characterization of UPEC isolates was performed by PCR-based phylotype analysis and serotyping. Nissle 1917 effects on growth inhibition and competitive overgrowth against UPEC isolates were evaluated in vitro using a plate-based competition assay. RESULTS: Feline phylogroups were similar to previous human and feline UPEC studies, with most of the isolates belonging to phylogroup A (42.5%), B2 (37.5%), and D (15.0%). Fifty-two percent of isolates were found to be resistant to antimicrobials, with 19% of these being multidrug resistant (MDR). Nissle 1917 adversely affected the growth of 82.5% of all isolates and 100% of MDR isolates in vitro. The median zone of inhibition was 3.33 mm (range, 1.67 to 10.67 mm). Thirteen isolates were affected via competitive overgrowth and 20 via growth inhibition. CLINICAL RELEVANCE: UPEC isolates from cats were similar in phylogroup analysis to human and dog isolates. The in vitro effects of Nissle 1917 on UPEC warrant additional studies to determine if similar results can be duplicated in vivo.

Escherichia coli pathotype contamination in raw canine diets.

OBJECTIVE: To investigate the prevalence of Escherichia coli contamination and E coli virulence gene signatures consistent with known E coli pathotypes in commercially available conventional diets and raw-meat-based diets (RMBDs). SAMPLE: 40 diets in total (19 conventionally cooked kibble or canned diets and 21 RMBDs) obtained from retail stores or online distributors. PROCEDURES: Each diet was cultured for E coli contamination in 3 separate container locations using standard microbiological techniques. Further characterization of E coli isolates was performed by polymerase chain reaction-based pathotype and virulence gene analysis. RESULTS: Conventional diets were negative in all culture based testing. In RMBDs, bacterial contamination was similar to previous reports in the veterinary literature, with 66% (14/21) of the RMBDs having positive cultures for E coli. Among the 191 confirmed E coli isolates from these diets, 31.9% (61/191) were positive for virulence genes. Categorized by pathotype, isolates presumptively belonging to the neonatal meningitis E coli pathotype (15.7% [30/191]) were the most common, followed by enterohemorrhagic E coli (10.5% [20/191]), enteropathogenic E coli (5.8% [11/191]), uropathogenic E coli (2.1% [4/191]), and diffusely adherent E coli (1.6% [3/191]). CLINICAL RELEVANCE: The results of this study reaffirmed the bacteriologic risks previously associated with RMBDs. Furthermore, potential zoonotic concerns associated with identified pathotypes in these diets may have significant consequences for owners in the animals' home environment. Potential risk associated with bacterial contamination should be addressed in animals fed RMBDs.

Suspected Primary Spontaneous Asymptomatic Pneumothorax in a Cat.

Spontaneous pneumothorax (SPT) is a documented emergency of the respiratory tract condition classified as either primary or secondary based on the presence of underlying pulmonary conditions. All reported SPT in the feline literature are evaluated for respiratory clinical signs. Primary SPT without underlying pathology or without clinical signs is not reported in cats. This case report describes a 10-year-old domestic longhair cat that was referred for evaluation of chronic lethargy with severe azotemia and placement of a subcutaneous ureteral bypass (SUB) system. Prior to presentation, the cat was diagnosed with renal insufficiency and treated medically with no resolution. Clinical examination under sedation revealed right-sided renomegaly. Thoracic radiographs revealed gas in the caudodorsal pleural space and concurrent pulmonary atelectasis. No respiratory clinical signs were present. Thoracic CT showed two pulmonary bullae, one located in the right caudal lung lobe and one in the cranial segment of the left cranial lung lobe. Abdominal ultrasound showed a right-sided ureteral obstruction. Medical management was elected for the spontaneous pneumothorax. A SUB was placed to address the ureteral obstruction; no complications were noted during recovery. The cat was free of clinical signs of respiratory disease after a follow-up time of nine months. This is the first reported case of a cat diagnosed with a nonclinical suspected primary spontaneous pneumothorax with no concurrent predisposing pulmonary pathology.

Synbiotic-IgY Therapy Modulates the Mucosal Microbiome and Inflammatory Indices in Dogs with Chronic Inflammatory Enteropathy: A Randomized, Double-Blind, Placebo-Controlled Study.

Chronic inflammatory enteropathy (CE) is a common cause of persistent gastrointestinal signs and intestinal inflammation in dogs. Since evidence links dysbiosis to mucosal inflammation, probiotics, prebiotics, or their combination (synbiotics) may reduce intestinal inflammation and ameliorate dysbiosis in affected dogs. This study's aim was to investigate the effects of the synbiotic-IgY supplement on clinical signs, inflammatory indices, and mucosal microbiota in dogs with CE. Dogs with CE were enrolled in a randomized prospective trial. Twenty-four client-owned dogs were fed a hydrolyzed diet and administered supplement or placebo (diet) for 6 weeks. Dogs were evaluated at diagnosis and 2- and 6-week post-treatment. Outcome measures included clinical activity, endoscopic and histologic scores, inflammatory markers (fecal calprotectin, C-reactive protein), and composition of the mucosal microbiota via FISH. Eleven supplement- and nine placebo-treated dogs completed the trial. After 6 weeks of therapy, clinical activity and endoscopic scores decreased in both groups. Compared to placebo-treated dogs, dogs administered supplement showed decreased calprotectin at 2-week post-treatment, decreased CRP at 2- and 6-week post-treatment increased mucosal Clostridia and Bacteroides and decreased Enterobacteriaceae in colonic biopsies at trial completion. Results suggest a beneficial effect of diet and supplements on host responses and mucosal microbiota in dogs with CE.

Secondary bile acid ursodeoxycholic acid alters weight, the gut microbiota, and the bile acid pool in conventional mice.

Ursodeoxycholic acid (commercially available as ursodiol) is a naturally occurring bile acid that is used to treat a variety of hepatic and gastrointestinal diseases. Ursodiol can modulate bile acid pools, which have the potential to alter the gut microbiota community structure. In turn, the gut microbial community can modulate bile acid pools, thus highlighting the interconnectedness of the gut microbiota-bile acid-host axis. Despite these interactions, it remains unclear if and how exogenously administered ursodiol shapes the gut microbial community structure and bile acid pool in conventional mice. This study aims to characterize how ursodiol alters the gastrointestinal ecosystem in conventional mice. C57BL/6J wildtype mice were given one of three doses of ursodiol (50, 150, or 450 mg/kg/day) by oral gavage for 21 days. Alterations in the gut microbiota and bile acids were examined including stool, ileal, and cecal content. Bile acids were also measured in serum. Significant weight loss was seen in mice treated with the low and high dose of ursodiol. Alterations in the microbial community structure and bile acid pool were seen in ileal and cecal content compared to pretreatment, and longitudinally in feces following the 21-day ursodiol treatment. In both ileal and cecal content, members of the Lachnospiraceae Family significantly contributed to the changes observed. This study is the first to provide a comprehensive view of how exogenously administered ursodiol shapes the healthy gastrointestinal ecosystem in conventional mice. Further studies to investigate how these changes in turn modify the host physiologic response are important.

Ursodeoxycholic Acid (UDCA) Mitigates the Host Inflammatory Response during Clostridioides difficile Infection by Altering Gut Bile Acids.

Clostridioides difficile infection (CDI) is associated with increasing morbidity and mortality posing an urgent threat to public health. Recurrence of CDI after successful treatment with antibiotics is high, thus necessitating discovery of novel therapeutics against this enteric pathogen. Administration of the secondary bile acid ursodeoxycholic acid (UDCA; ursodiol) inhibits the life cycles of various strains of C. difficilein vitro, suggesting that the FDA-approved formulation of UDCA, known as ursodiol, may be able to restore colonization resistance against C. difficilein vivo However, the mechanism(s) by which ursodiol is able to restore colonization resistance against C. difficile remains unknown. Here, we confirmed that ursodiol inhibits C. difficile R20291 spore germination and outgrowth, growth, and toxin activity in a dose-dependent manner in vitro In a murine model of CDI, exogenous administration of ursodiol resulted in significant alterations in the bile acid metabolome with little to no changes in gut microbial community structure. Ursodiol pretreatment resulted in attenuation of CDI pathogenesis early in the course of disease, which coincided with alterations in the cecal and colonic inflammatory transcriptome, bile acid-activated receptors nuclear farnesoid X receptor (FXR) and transmembrane G-protein-coupled membrane receptor 5 (TGR5), which are able to modulate the innate immune response through signaling pathways such as NF-κB. Although ursodiol pretreatment did not result in a consistent decrease in the C. difficile life cycle in vivo, it was able to attenuate an overly robust inflammatory response that is detrimental to the host during CDI. Ursodiol remains a viable nonantibiotic treatment and/or prevention strategy against CDI. Likewise, modulation of the host innate immune response via bile acid-activated receptors FXR and TGR5 represents a new potential treatment strategy for patients with CDI.

Diversification of host bile acids by members of the gut microbiota.

Bile acid biotransformation is a collaborative effort by the host and the gut microbiome. Host hepatocytes synthesize primary bile acids from cholesterol. Once these host-derived primary bile acids enter the gastrointestinal tract, the gut microbiota chemically modify them into secondary bile acids. Interest into the gut-bile acid-host axis is expanding in diverse fields including gastroenterology, endocrinology, oncology, and infectious disease. This review aims to 1) describe the physiologic aspects of collaborative bile acid metabolism by the host and gut microbiota; 2) to evaluate how gut microbes influence bile acid pools, and in turn how bile acid pools modulate the gut microbial community structure; 3) to compare species differences in bile acid pools; and lastly, 4) discuss the effects of ursodeoxycholic acid (UDCA) administration, a common therapeutic bile acid, on the gut microbiota-bile acid-host axis.

Inhibition of spore germination, growth, and toxin activity of clinically relevant C. difficile strains by gut microbiota derived secondary bile acids.

The changing epidemiology of Clostridium difficile infection over the past decades presents a significant challenge in the management of C. difficile associated diseases. The gastrointestinal tract microbiota provides colonization resistance against C. difficile, and growing evidence suggests that gut microbial derived secondary bile acids (SBAs) play a role. We hypothesized that the C. difficile life cycle; spore germination and outgrowth, growth, and toxin production, of strains that vary by age and ribotype will differ in their sensitivity to SBAs. C. difficile strains R20291 and CD196 (ribotype 027), M68 and CF5 (017), 630 (012), BI9 (001) and M120 (078) were used to define taurocholate (TCA) mediated spore germination and outgrowth, growth, and toxin activity in the absence and presence of gut microbial derived SBAs (deoxycholate, isodeoxycholate, lithocholate, isolithocholate, ursodeoxycholate, ω-muricholate, and hyodeoxycholate) found in the human and mouse large intestine. C. difficile strains varied in their rates of germination, growth kinetics, and toxin activity without the addition of SBAs. C. difficile M120, a highly divergent strain, had robust germination, growth, but significantly lower toxin activity compared to other strains. Many SBAs were able to inhibit TCA mediated spore germination and outgrowth, growth, and toxin activity in a dose dependent manner, but the level of inhibition and resistance varied across all strains and ribotypes. This study illustrates how clinically relevant C. difficile strains can have different responses when exposed to SBAs present in the gastrointestinal tract.

Impact of microbial derived secondary bile acids on colonization resistance against Clostridium difficile in the gastrointestinal tract.

Clostridium difficile is an anaerobic, Gram positive, spore-forming bacillus that is the leading cause of nosocomial gastroenteritis. Clostridium difficile infection (CDI) is associated with increasing morbidity and mortality, consequently posing an urgent threat to public health. Recurrence of CDI after successful treatment with antibiotics is high, thus necessitating discovery of novel therapeutics against this pathogen. Susceptibility to CDI is associated with alterations in the gut microbiota composition and bile acid metabolome, specifically a loss of microbial derived secondary bile acids. This review aims to summarize in vitro, ex vivo, and in vivo studies done by our group and others that demonstrate how secondary bile acids affect the different stages of the C. difficile life cycle. Understanding the dynamic interplay of C. difficile and microbial derived secondary bile acids within the gastrointestinal tract will shed light on how bile acids play a role in colonization resistance against C. difficile. Rational manipulation of secondary bile acids may prove beneficial as a treatment for patients with CDI.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291.

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.