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Protozoal diarrhea caused by Tritrichomonas foetus (blagburni) is a prevalent, lifelong, and globally distributed burden in domestic cats. Treatment is limited to the use of 5-nitroimidazoles and treatment failure is common. The repurposed gold salt compound auranofin has killing activity against diverse protozoa in vitro but evidence of efficacy in naturally occurring protozoal infections is lacking. This exploratory study investigated the efficacy and safety of auranofin for treatment of cats with naturally occurring, 5-nitroimidazole-resistant, T. foetus infection. The minimum lethal concentration (MLC) of auranofin against 5 isolates of feline T. foetus was determined under aerobic conditions in vitro. Healthy cats and cats with T. foetus infection were treated with immediate release auranofin (range, 0.5-3 mg/cat for 7 days) or guar gum-coated auranofin capsules (0.5 or 3 mg/cat for 7 days). Adverse effects were monitored by clinical signs and clinicopathologic testing. Efficacy was determined by fecal consistency score, bowel movement frequency, and single-tube nested PCR of feces for T. foetus rDNA. Fecal samples were assayed for concentrations of auranofin, known and predicted metabolites of auranofin, gold containing molecules, and total gold content using HPLC, LC-MS, ion mobility-MS, and ICP-MS, respectively. Auranofin was effective at killing isolates of feline T. foetus at MLC ≥ 1 μg/ml. Treatment of cats with T. foetus infection with either immediate release auranofin or a colon-targeted guar gum-coated tablet of auranofin did not eradicate infection. Treatment failure occurred despite fecal concentrations of gold that met or exceeded the equivalent MLC of auranofin. Neither auranofin, known or predicted metabolites of auranofin, nor any gold-containing molecules >100 Da could be detected in fecal samples of treated cats. Adverse effects associated with auranofin treatment were common but minor. These studies identify that in vitro susceptibility test results of auranofin may not translate to treatment effectiveness in vivo even when achieving gold concentrations equivalent to the MLC of auranofin in the target environment. These studies further establish the absence of any predicted or unpredicted gold containing metabolites in feces after oral administration of auranofin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mucocele formation in dogs is a unique and enigmatic muco-obstructive disease of the gallbladder caused by the amassment of abnormal mucus that bears striking pathological similarity to cystic fibrosis. We investigated the role of cystic fibrosis transmembrane conductance regulatory protein (CFTR) in the pathogenesis of this disease. The location and frequency of disease-associated variants in the coding region of CFTR were compared using whole genome sequence data from 2,642 dogs representing breeds at low-risk, high-risk, or with confirmed disease. Expression, localization, and ion transport activity of CFTR were quantified in control and mucocele gallbladders by NanoString, Western blotting, immunofluorescence imaging, and studies in Ussing chambers. Our results establish a significant loss of CFTR-dependent anion secretion by mucocele gallbladder mucosa. A significantly lower quantity of CFTR protein was demonstrated relative to E-cadherin in mucocele compared with control gallbladder mucosa. Immunofluorescence identified CFTR along the apical membrane of epithelial cells in control gallbladders but not in mucocele gallbladder epithelium. Decreases in mRNA copy number for CFTR were accompanied by decreases in mRNA for the Cl - /[Formula: see text] exchanger SLC26A3 , K + channels ( KCNQ1 , KCNN4 ), and vasoactive intestinal polypeptide receptor ( VIPR1 ), which suggest a driving force for change in secretory function of gallbladder epithelial cells in the pathogenesis of mucocele formation. There were no significant differences in CFTR gene variant frequency, type, or predicted impact comparing low-risk, high-risk, and definitively diagnosed groups of dogs. This study describes a unique, naturally occurring muco-obstructive disease of the canine gallbladder, with uncanny similarity to cystic fibrosis, and driven by the underlying failure of CFTR function. NEW & NOTEWORTHY Cystic fibrosis transmembrane conductance regulatory protein (CFTR) genomic variants and expression of mRNA, protein, and electrogenic anion secretory activity of CFTR were characterized in dog gallbladder. Acquired inhibition of CFTR expression by gallbladder epithelium was identified as underpinning a naturally occurring muco-obstructive disease of the dog gallbladder that bears striking pathological similarity to animal models of cystic fibrosis.

Background: Gallbladder disease in people is frequently associated with disorders of lipid metabolism and metabolic syndrome. A recently emergent gallbladder disease of dogs, referred to as mucocele formation, is characterized by secretion of abnormal mucus by the gallbladder epithelium and is similarly associated with hyperlipidemia, endocrinopathy, and metabolic dysfunction. The cause of gallbladder mucocele formation in dogs is unknown., Methods: A prospective case-controlled study was conducted to gain insight into disease pathogenesis by characterization of plasma lipid abnormalities in 18 dogs with gallbladder mucocele formation and 18 age and breed matched control dogs using direct infusion mass spectrometry for complex plasma lipid analysis. This analysis was complemented by histochemical and ultrastructural examination of gallbladder mucosa from dogs with gallbladder mucocele formation and control dogs for evidence of altered lipid homeostasis of the gallbladder epithelium., Results: Gallbladder mucocele formation in dogs carried a unique lipidomic signature of increased lipogenesis impacting 50% of lipid classes, 36% of esterified fatty acid species, and 11% of complex lipid species. Broad enrichment of complex lipids with palmitoleic acid (16:1) and decreased abundance within complex lipids of presumptive omega-3 fatty acids eicosapentaenoic (20:5) and docosahexaenoic (22:6) was significant. Severe lipidosis of gallbladder epithelium pinpoints the gallbladder as involved causally or consequently in abnormal lipid metabolism., Conclusion: Our study supports a primary increase in lipogenesis in dogs with mucocele formation and abnormal gallbladder lipid metabolism in disease pathogenesis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gookin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Conjoining of the major pancreatic duct and common bile duct at the major duodenal papilla (MDP) is suspected to predispose cats to the clinical syndrome of "triaditis." However, microanatomy of the MDP or presence of lesions at the MDP has not been assessed in cats with or without triaditis. The aims of this study were to characterize feline MDP histomorphology and to identify associations between MDP anatomy/disease and the presence of biliary, pancreatic, or intestinal inflammation or neoplasia. Histologic assessment was prospectively performed on the MDP, duodenum, jejunum, ileum, liver, and pancreas from 124 client-owned cats undergoing postmortem examination. The majority of cats (104/124, 84%) had a complex ductular network at the MDP, with no distinction between pancreatic and common bile ducts. Lymphoid aggregates at the MDP were common (63/124, 51%). Inflammation of the MDP (MDPitis) was present in 35 of 124 cats (28%) and was often concurrent with cholangitis, pancreatitis, or enteritis (32/35, 91%), but was only associated with enteritis (19/35, 54%, P < .05). Triaditis was less common (19/124, 15%), but was associated with both conjoined MDP anatomy (19/19, 100%, P < .05) and MDPitis (12/19, 63%, P < .05). Neoplasia was present in 37 of 124 cats (29%), with lymphoma (28/37, 78%) predominating. Enteropathy-associated T-cell lymphoma type 2 (EATL2) was most common ( n = 16/37, 43%) and was associated with triaditis and MDPitis ( P < .05). These findings suggest that anatomy, immune activation, and/or inflammation of the MDP may play a role in the pathogenesis of triaditis. Further studies are needed to elucidate the relationships between triaditis, MDPitis, and EATL2., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Bacterial infection of bile is a common cause of hepatobiliary disease in cats. Whether bile harbors a core microbiota in health or in cases of suspected hepatobiliary disease in cats is unknown., Objectives: Establish if gallbladder bile in apparently healthy cats harbors a core microbiota composed of bacterial taxa common to many individuals. Compare results of bile cytology, bile culture, and 16S rRNA gene amplicon sequencing in apparently healthy cats and cats with suspected hepatobiliary disease., Animals: Forty-three client-owned cats with suspected hepatobiliary disease and 17 control cats., Methods: Bile was collected by ultrasound guided cholecystocentesis (cats with suspected hepatobiliary disease) or laparotomy after euthanasia (controls). Bile samples underwent cytologic examination, aerobic and anaerobic culture, and DNA was extracted for 16S rRNA gene amplification and sequencing., Results: Microbiome sequencing did not identify a core microbiota in control cats or cats having bile sampled because of clinical suspicion for hepatobiliary disease. Microbiome profiles from control cats were indistinguishable from profiles obtained from sampling instruments and reagents that were not exposed to bile (technical controls). Bacterial taxa that could not be explained by contamination or off-target amplification were identified only in samples from cats with bactibilia and positive bile culture results for Escherichia coli. In several E. coli positive samples, microbiome sequencing also identified a small number of potentially co-infecting bacterial genera not identified by culture., Conclusions and Clinical Importance: Cat bile does not harbor a core microbiota. Uncultured bacteria may contribute to pathogenesis of hepatobiliary disease in cats with bile E. coli infection., (© 2023 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

To date studies have not investigated the culture-independent microbiome of bile from dogs, a species where aseptic collection of bile under ultrasound guidance is somewhat routine. Despite frequent collection of bile for culture-based diagnosis of bacterial cholecystitis, it is unknown whether bile from healthy dogs harbors uncultivable bacteria or a core microbiota. The answer to this question is critical to understanding the pathogenesis of biliary infection and as a baseline to exploration of other biliary diseases in dogs where uncultivable bacteria could play a pathogenic role. A pressing example of such a disease would be gallbladder mucocele formation in dogs. This prevalent and deadly condition is characterized by excessive secretion of abnormal mucus by the gallbladder epithelium that can eventually lead to rupture of the gallbladder or obstruction of bile flow. The cause of mucocele formation is unknown as is whether uncultivable, and therefore unrecognized, bacteria play any systematic role in pathogenesis. In this study we applied next-generation 16S rRNA gene sequencing to identify the culture-negative bacterial community of gallbladder bile from healthy dogs and gallbladder mucus from dogs with mucocele formation. Integral to our study was the use of 2 separate DNA isolations on each sample using different extraction methods and sequencing of negative control samples enabling recognition and curation of contaminating sequences. Microbiota findings were validated by simultaneous culture-based identification, cytological examination of bile, and fluorescence in-situ hybridization (FISH) performed on gallbladder mucosa. Using culture-dependent, cytological, FISH, and 16S rRNA sequencing approaches, results of our study do not support existence of a core microbiome in the bile of healthy dogs or gallbladder mucus from dogs with mucocele formation. Our findings further document how contaminating sequences can significantly contribute to the results of sequencing analysis when performed on samples with low bacterial biomass., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Gookin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Diarrhea is the second most common cause of mortality in shelter kittens. Studies examining prevention strategies in this population are lacking. Probiotics are of particular interest but studies in cats are largely limited to healthy adults or those with induced disease. Only one study in domestic cats describes the use of host-derived bacteria as a probiotic. We previously identified Enterococcus hirae as a dominant species colonizing the small intestinal mucosa in healthy shelter kittens. Oral administration of a probiotic formulation of kitten-origin E. hirae (strain 1002-2) mitigated the increase in intestinal permeability and fecal water loss resulting from experimental enteropathogenic E. coli infection in purpose-bred kittens. Based on these findings, we hypothesized that administration of kitten-origin E. hirae to weaned fostered shelter kittens could provide a measurable preventative health benefit., Methods: We conducted a randomized, placebo-controlled, blinded clinical trial to determine the impact of a freeze-dried E. hirae probiotic on body weight gain, incidence of diarrhea, carriage of potential diarrheal pathogens, and composition of the intestinal microbiota in weaned fostered shelter kittens., Results: One-hundred thirty kittens completed the study. Fifty-eight kittens received the probiotic and 72 received the placebo. There were no significant differences in age, weight upon initiation of the study, number of days in the study, average daily gain in body weight, or weight at completion of the study. Kittens treated with E. hirae were 3.4 times less likely to develop diarrhea compared to kittens treated with placebo (odds ratio = 0.294, 95% CI 0.109-0.792, p = 0.022). A significant impact of E. hirae was not observed on the presence or abundance of 30 different bacterial, viral, protozoal, fungal, algal, and parasitic agents in feces examined by qPCR. With exception to a decrease in Megamonas , administration of the E. hirae probiotic did not alter the predominant bacterial phyla present in feces based on 16S rRNA gene amplicon sequencing., Discussion: Decreased incidence of diarrhea associated with preventative administration of E. hirae to foster kittens supports a rationale for use of E. hirae for disease prevention in this young population at high risk for intestinal disease though additional studies are warranted., Competing Interests: Authors ME and AS were employed by IDEXX Laboratories, Inc. Author JB was employed by Vet Med Labor GmbH Division, IDEXX Laboratories, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gookin, Strong, Bruno-Bárcena, Stauffer, Williams, Wassack, Azcarate-Peril, Estrada, Seguin, Balzer and Davidson.)

Background: Proteinuria is an independent risk factor for morbidity and mortality in dogs. An association between proteinuria and gallbladder mucocele formation in dogs is unknown., Objective: Determine if gallbladder mucocele formation or clinicopathologic comorbidities are associated with proteinuria., Animals: Twenty-five dogs with mucocele formation and 25 breed and age-matched control dogs from a prior study., Methods: Retrospective case control study. Proteinuria defined by calculated urine dipstick protein concentration (mg/mL) to urine specific gravity (USG) ratio. Clinicopathologic findings, postcosyntropin cortisol concentration, thyroid function profile, and illness severity score were recorded., Results: Median urine dipstick protein concentration to USG ratio and number of dogs having a ratio ≥1.5 were significantly higher for dogs with mucocele formation compared to control dogs. Proteinuria was not significantly associated with CBC or serum biochemistry profile abnormalities but increased in relation to severity of illness., Conclusions and Clinical Importance: Gallbladder mucocele formation is significantly associated with proteinuria in dogs. Diagnosis and treatment of proteinuria in dogs with mucocele formation might minimize long term kidney morbidity in these patients., (© 2021 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC. on behalf of the American College of Veterinary Internal Medicine.)

The protozoal pathogen Tritrichomonas foetus infects the colon of domestic cats and is a major cause of chronic colitis and diarrhea. Treatment failure is common, but antibiotics may improve clinical signs in a subset of cats, leading researchers to question involvement of the colonic microbiota in disease pathogenesis. Studies performed in women with venereal Trichomonas vaginalis infections have revealed that dysbiosis of host microbiota contributes to pathogenicity with similar findings also found in mice with intestinal Tritrichomonas musculis The aim of this study was to characterize differences in the fecal microbiota of cats with and without naturally occurring T. foetus infection and in a group of kittens prior to and after experimentally induced infection. Archived fecal DNA from cats undergoing testing for T. foetus infection (n = 89) and experimentally infected kittens (n = 4; at pre-, 2 weeks, and 9 weeks post-infection) were analyzed by sequencing of 16S rRNA genes. Amongst the naturally infected population, the genera Megamonas and Helicobacter were significantly increased in prevalence and abundance in cats testing positive for T. foetus infection. In the group of four experimentally infected kittens, fecal samples post-infection had significantly lower abundance of genus Dialister and Megamonas and greater abundance of the class Betaproteobacteria and family Succinivibrionaceae. We hypothesize that T. foetus promotes dysbiosis by competition for fermentable substrates used by these bacteria and that metabolic byproducts may contribute to the pathogenesis of colonic inflammation and diarrhea. Future studies are warranted for the measurement of fecal concentrations of microbial and protozoal metabolites in cats with T. foetus infection for the identification of potential therapeutic targets., Competing Interests: The authors have declared that no competing interests exist.

Typical enteropathogenic Escherichia coli (tEPEC) is a leading cause of diarrhea and associated death in children worldwide. Atypical EPEC (aEPEC) lacks the plasmid encoding bundle-forming pili and is considered less virulent, but the molecular mechanism of virulence is poorly understood. We recently identified kittens as a host for aEPEC where intestinal epithelial colonization was associated with diarrheal disease and death. The purposes of this study were to (i) determine the genomic similarity between kitten aEPEC and human aEPEC isolates and (ii) identify genotypic or phenotypic traits associated with virulence in kitten aEPEC. We observed no differences between kitten and human aEPEC in core genome content or gene cluster sequence identities, and no distinguishing genomic content was observed between aEPEC isolates from kittens with nonclinical colonization (NC) versus those with lethal infection (LI). Variation in adherence patterns and ability to aggregate actin in cultured cells mirrored descriptions of human aEPEC. The aEPEC isolated from kittens with LI were significantly more motile than isolates from kittens with NC. Kittens may serve as a reservoir for aEPEC that is indistinguishable from human aEPEC isolates and may provide a needed comparative animal model for the study of aEPEC pathogenesis. Motility seems to be an important factor in pathogenesis of LI associated with aEPEC in kittens., (Copyright © 2021 American Society for Microbiology.)

Objectives: The objective of this study was to identify significant associations between treatment interventions and survival of orphaned shelter kittens with diarrhea., Methods: Orphaned kittens admitted to a large open-intake municipal animal shelter and entering a volunteer foster care provider network between April 2016 and July 2017 were studied. Individual orphaned kittens for which the care provider sought examination by shelter veterinarians because of clinical signs of diarrhea were included. Treatments administered were recorded and their association with kitten survival to adoption or transfer to a rescue partner was statistically examined., Results: A total of 1718 orphaned kittens were fostered, among which 220 kittens (12.8%) from 118 different litters were presented for evaluation of diarrhea. A total of 172 (78.2%) kittens underwent treatment for their diarrhea, among which 153 (89.0%) survived to adoption or transfer to a rescue partner and 19 (11.0%) died or were humanely euthanized. Kittens with diarrhea that were ⩾4 weeks of age were 24.8 times more likely to survive (95% confidence interval [CI] 6.8-89.8; P <0.0001). While controlling for age, kittens that received a vitamin and mineral supplement were 12.8 times more likely to survive (95% CI 3.1-52.5; P = 0.0004) than kittens with diarrhea that did not receive the supplement. Treatment with subcutaneous fluids, penicillin G, tube feeding, a probiotic containing Enterococcus faecium SF68, ponazuril or metronidazole did not statistically significantly increase the survival of kittens with diarrhea., Conclusions and Relevance: Age ⩾4 weeks and treatment of diarrhea with a vitamin and mineral supplement favorably improves survival of orphaned kittens to adoption. Application of these findings are likely to improve the health and welfare of this population and contribute to a refinement in use of shelter resources.

Background: Fecal polymerase chain reaction (PCR) testing for Tritrichomonas foetus is considered the most sensitive means for diagnosis of infection but results could be influenced by fecal collection technique and prior use of antimicrobial drugs., Objectives: To establish any association between fecal collection technique or treatment history and results of fecal PCR testing for T. foetus., Animals: Fecal samples from 1717 cats submitted by veterinarians between January 2012 and December 2017., Methods: This study used a retrospective analysis. T. foetus PCR test results from 1808 fecal samples submitted for diagnostic testing were examined for their association with method of fecal collection and prior antimicrobial treatments. Data were collected from sample submission form., Results: Positive T. foetus PCR test results were obtained for 274 (16%) cats. Fecal samples collected via fecal loop had increased probability of positive PCR test results (odds ratio [OR] 2.04, 95% confidence interval [CI] 1.31-3.17, P = .002) compared to samples collected by colonic flush. There was no association between PCR test results and treatment history, treatment type, or prior treatment with ronidazole. After an initial positive PCR test, 4/19 (21%; 95% CI 2.7%-39.4%) cats treated with ronidazole had a second positive test result., Conclusions and Clinical Importance: Results of this study support that fecal samples collected by loop might be better for PCR diagnosis of T. foetus infection. Lack of association of ronidazole with PCR test results and a 21% all-potential-causes failure rate of ronidazole in cats with preconfirmed infection are important limitations to use of this drug., (© 2020 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: The publication requirement for board certification in Small Animal Internal Medicine (SAIM) by the ACVIM is controversial., Objectives: Directly and indirectly evaluate the perceptions SAIM Diplomates have on the publication requirement. A secondary objective was to compare the frequency with which publications submitted for credentialing purposes (CredPubs) were cited compared to control articles., Subjects: One thousand two hundred forty-one SAIM Diplomates were sent an electronic survey., Methods: A electronic survey was sent to all SAIM Diplomates. Practice websites were evaluated for reference to publication or research. An electronic database was searched to identify the number of times a subset of CredPubs were cited was compared to control articles., Results: Five hundred six individuals responded. The majority of respondents (n = 428, 85.25%) stated the requirement should be retained either with no changes (n = 186, 37.05%) or with clarifications or modifications (n = 242, 48.21%). A minority of respondents (n = 74, 14.7%) felt it should be eliminated. "Understanding the scientific process" was the most commonly selected reason (n = 467, 92.48%) for the publication requirement. All websites that mentioned research or publication did so using a positive sentiment. With regard to relative citation rates; 17% of CredPubs were in the lower quartile, 59.1% of CredPubs were in the interquartile range, and 23.5% were in the upper quartile compared to control articles., Conclusions and Clinical Importance: A majority of SAIM Diplomates favored the retention of the publication requirement in some form. CredPubs were cited at rates similar to control articles., (© 2020 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Tritrichomonas foetus is a common cause of large bowel diarrhea in cats. Probiotics have been suggested to be effective for many intestinal pathogens; however, there are a lack of studies evaluating the effect of probiotics in T. foetus infection. In vitro studies were performed to evaluate the effect of a probiotic containing Enterococcus faecium (Efm) SF68 and a novel probiotic, Enterococcus hirae, on the inhibition of T. foetus growth, adhesion to, and cytotoxicity towards the intestinal epithelium. The effect of enterococci on T. foetus proliferation during co-culture was evaluated throughout log phase T. foetus growth. The previously validated in vitro co-culture model system using porcine intestinal epithelial cells (IPEC-J2) was used to evaluate the effect of enterococci on T. foetus adhesion and cytotoxicity towards intestinal epithelial cells. Cytotoxicity was assessed using fluorescent microscopy and spectrophotometry. Interactions of T. foetus, enterococci, and intestinal epithelial cells were assessed using scanning electron microscopy and immunofluorescence assays (IFA). Enterococcus-induced inhibition of T. foetus growth was demonstrated at concentrations as low as 10 4 enterococci colony forming units (CFU)/mL and was dependent, in part, on environmental pH and the presence of viable enterococci organisms. T. foetus adhesion, including with a ronidazole-resistant strain, was reduced with pretreatment of intestinal epithelial cells with enterococci but was not significantly affected when enterococci were introduced simultaneously or following T. foetus infection. Compared to Efm, E. hirae more effectively decreased T. foetus adhesion, suggesting its superior potential as a novel probiotic for T. foetus infection. There was no effect of enterococci treatment on T. foetus-induced intestinal epithelial cell cytotoxicity. Our results support further study into the investigation of a possible benefit of enterococci-containing probiotic treatment for prevention of T. foetus infection in at-risk uninfected cats., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Typical enteropathogenic E. coli (tEPEC) carries the highest hazard of death in children with diarrhea and atypical EPEC (aEPEC) was recently identified as significantly associated with diarrheal mortality in kittens. In both children and kittens there is a significant association between aEPEC burden and diarrheal disease, however the infection can be found in individuals with and without diarrhea. It remains unclear to what extent, under what conditions, or by what mechanisms aEPEC serves as a primary pathogen in individuals with diarrhea. It seems likely that a combination of host and bacterial factors enable aEPEC to cause disease in some individuals and not in others. The purpose of this study was to determine the impact of aEPEC on intestinal function and diarrhea in kittens following experimentally-induced carriage and the influence of a disrupted intestinal microbiota on disease susceptibility. Results of this study identify aEPEC as a potential pathogen in kittens. In the absence of disruption to the intestinal microbiota, kittens are resistant to clinical signs of aEPEC carriage but demonstrate significant occult changes in intestinal absorption and permeability. Antibiotic-induced disruption of the intestinal microbiota prior to infection increases subsequent intestinal water loss as determined by % fecal wet weight. Enrichment of the intestinal microbiota with a commensal member of the feline mucosa-associated microbiota, Enterococcus hirae, ameliorated the effects of aEPEC experimental infection on intestinal function and water loss. These observations begin to unravel the mechanisms by which aEPEC infection may be able to exploit susceptible hosts., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Gallbladder mucocele formation is an emerging disease in dogs characterized by increased secretion of condensed granules of gel-forming mucin by the gallbladder epithelium and formation of an abnormally thick mucus that can culminate in obstruction of the bile duct or rupture of the gallbladder. The disease is associated with a high morbidity and mortality and its pathogenesis is unknown. Affected dogs have a significantly increased likelihood of concurrent diagnosis of hyperadrenocorticism, hypothyroidism, and hyperlipidemia. Whether these endocrinopathies represent coincidental primary disease processes that exacerbate gallbladder mucocele formation in predisposed dogs or reflect a concurrent disruption of endocrine and lipid metabolism is unclear. In this study, we investigated a hypothesis that dogs with gallbladder mucocele formation would have a high prevalence of occult and atypical abnormalities in adrenal cortical and thyroid gland function that would suggest the presence of endocrine disruption and provide deeper insight into disease pathogenesis. We performed a case-control study of dogs with and without ultrasonographic diagnosis of gallbladder mucocele formation and profiled adrenal cortical function using a quantitative mass spectrometry-based assay of serum adrenal-origin steroids before and after administration of synthetic cosyntropin. We simultaneously profiled serum thyroid hormone concentrations and evaluated iodine sufficiency by measurement of urine iodine:creatinine ratios (UICR). The studies were complemented by histological examination of archival thyroid tissue and measurements of thyroid gland organic iodine from dogs with gallbladder mucocele formation and control dogs. Dogs with gallbladder mucocele formation demonstrated an exaggerated cortisol response to adrenal stimulation with cosyntropin. A prevalence of 10% of dogs with gallbladder mucocele formation met laboratory-based criteria for suspect or definitive diagnosis of hyperadrenocorticism. A significantly greater number of dogs with gallbladder mucocele formation had basal serum dehydroepiandrosterone (DHEAS) increases compared to control dogs. A high percentage of dogs with gallbladder mucocele formation (26%) met laboratory-based criteria for diagnosis of hypothyroidism, but lacked detection of anti-thyroglobulin antibodies. Dogs with gallbladder mucocele formation had significantly higher UICRs than control dogs. Examination of thyroid tissue from an unrelated group of dogs with gallbladder mucocele formation did not demonstrate histological evidence of thyroiditis or significant differences in content of organic iodine. These findings suggest that dogs with gallbladder mucocele formation have a greater capacity for cortisol synthesis and pinpoint DHEAS elevations as a potential clue to the underlying pathogenesis of the disease. A high prevalence of thyroid dysfunction with absent evidence for autoimmune thyroiditis suggest a disrupted thyroid hormone metabolism in dogs with gallbladder mucocele formation although an influence of non-thyroidal illness cannot be excluded. High UICR in dogs with gallbladder mucocele formation is of undetermined significance, but of interest for further study., Competing Interests: The authors have declared that no competing interests exist.

Background & Aims: The epithelial response is critical for intestinal defense against Cryptosporidium, but is poorly understood. To uncover the host strategy for defense against Cryptosporidium, we examined the transcriptional response of intestinal epithelial cells (IECs) to C parvum in experimentally infected piglets by microarray. Up-regulated genes were dominated by targets of interferon (IFN) and IFN-λ3 was up-regulated significantly in infected piglet mucosa. Although IFN-λ has been described as a mediator of epithelial defense against viral pathogens, there is limited knowledge of any role against nonviral pathogens. Accordingly, the aim of the study was to determine the significance of IFN-λ3 to epithelial defense and barrier function during C parvum infection., Methods: The significance of C parvum-induced IFN-λ3 expression was determined using an immunoneutralization approach in neonatal C57BL/6 mice. The ability of the intestinal epithelium to up-regulate IFN-λ2/3 expression in response to C parvum infection and the influence of IFN-λ2/3 on epithelial defense against C parvum invasion, intracellular development, and loss of barrier function was examined using polarized monolayers of a nontransformed porcine-derived small intestinal epithelial cell line (IPEC-J2). Specifically, changes in barrier function were quantified by measurement of transepithelial electrical resistance and transepithelial flux studies., Results: Immunoneutralization of IFN-λ2/3 in C parvum-infected neonatal mice resulted in a significantly increased parasite burden, fecal shedding, and villus blunting with crypt hyperplasia during peak infection. In vitro, C parvum was sufficient to induce autonomous IFN-λ3 and interferon-stimulated gene 15 expression by IECs. Priming of IECs with recombinant human IFN-λ3 promoted cellular defense against C parvum infection and abrogated C parvum-induced loss of barrier function by decreasing paracellular permeability to sodium., Conclusions: These studies identify IFN-λ3 as a key epithelial defense mechanism against C parvum infection., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Mucocele formation is characterized by secretion of abnormally thick mucus by the gallbladder epithelium of dogs that may cause obstruction of the bile duct or rupture of the gallbladder. The disease is increasingly recognized and is associated with a high morbidity and mortality. The cause of gallbladder mucocele formation in dogs is unknown. There is a strong breed predisposition and affected dogs have a high incidence of concurrent endocrinopathy or hyperlipidemia. These observations suggest a significant influence of both genetic and metabolic factors on disease pathogenesis. In this study, we investigated a theory that mucocele formation is associated with a syndrome of metabolic disruption. We surmised that a global, untargeted metabolomics approach could provide unique insight into the systemic pathogenesis of gallbladder mucocele formation and identify specific compounds as candidate biomarkers or treatment targets. Moreover, concurrent examination of the serum and hepatic duct bile metabolome would enable the construction of mechanism-based theories or identification of specific compounds responsible for altered function of the gallbladder epithelium. Abnormalities observed in dogs with gallbladder mucocele formation, including a 33-fold decrease in serum adenosine 5'-monophosphate (AMP), lower quantities of precursors required for synthesis of energy transporting nucleotides, and increases in citric acid cycle intermediates, suggest excess metabolic energy and a carbon surplus. Altered quantities of compounds involved in protein translation and RNA turnover, together with accumulation of gamma-glutamylated and N-acetylated amino acids in serum suggest abnormal regulation of protein and amino acid metabolism. Increases in lathosterol and 7α-hydroxycholesterol suggest a primary increase in cholesterol synthesis and diversion to bile acid formation. A number of specific biomarker compounds were identified for their ability to distinguish between control dogs and those that formed a gallbladder mucocele. Particularly noteworthy was a significant decrease in quantity of biologically active compounds that stimulate biliary ductal fluid secretion including adenosine, cAMP, taurolithocholic acid, and taurocholic acid. These findings support the presence of significant metabolic disruption in dogs with mucocele formation. A targeted, quantitative analysis of the identified serum biomarkers is warranted to determine their utility for diagnosis of this disease. Finally, repletion of compounds whose biological activity normally promotes biliary ductal secretion should be examined for any therapeutic impact for resolution or prevention of mucocele formation.

Background: Bacterial cholecystitis often is diagnosed by combination of gallbladder ultrasound (US) findings and positive results of bile culture. The value of gallbladder US in determining the likelihood of bile bacterial infection in cats and dogs with suspected biliary disease is unknown., Hypothesis/objectives: To determine the value of gallbladder US in predicting bile bacterial culture results, identify most common bacterial isolates from bile, and describe complications after cholecystocentesis in cats and dogs with suspected hepatobiliary disease., Animals: Cats (70) and dogs (202) that underwent an abdominal US and submission of bile for culture were included in the study., Methods: A cross-sectional study design was used to determine the association of gallbladder US abnormalities and the results of bile cultures, and complications of cholecystocentesis., Results: Abnormal gallbladder US had high sensitivity (96%) but low specificity (49%) in cats with positive and negative results of bile bacterial culture, respectively. Cats with normal gallbladder US findings were unlikely to have positive bile bacterial culture (negative predictive value of 96%). Gallbladder US had lower sensitivity (81%), specificity (31%), positive predictive value (20%), and negative predictive value (88%) in dogs. The most common bacterial isolates were of enteric origin, the prevalence being higher in cats. Incidence of complications after cholecystocentesis was 3.4%., Conclusions and Clinical Importance: Gallbladder US has a high negative predictive value for bile culture results in cats. This modality is less predictive of infection in dogs. Percutaneous US-guided cholecystocentesis has a low complication rate., (Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Diarrhea is responsible for the death of approximately 900,000 children per year worldwide. In children, typical enteropathogenic Escherichia coli (EPEC) is a common cause of diarrhea and is associated with a higher hazard of death. Typical EPEC infection is rare in animals and poorly reproduced in experimental animal models. In contrast, atypical EPEC (aEPEC) infection is common in both children and animals, but its role in diarrhea is uncertain. Mortality in kittens is often attributed to diarrhea, and we previously identified enteroadherent EPEC in the intestines of deceased kittens. The purpose of this study was to determine the prevalence and type of EPEC in kittens and whether infection was associated with diarrhea, diarrhea-related mortality, gastrointestinal pathology, or other risk factors. Kittens with and without diarrhea were obtained from two shelter facilities and determined to shed atypical EPEC at a culture-based prevalence of 18%. In contrast, quantitative PCR detected the presence of the gene for intimin ( eae ) in feces from 42% of kittens. aEPEC was isolated from kittens with and without diarrhea. However, kittens with diarrhea harbored significantly larger quantities of aEPEC than kittens without diarrhea. Kittens with aEPEC had a significantly greater severity of small intestinal and colonic lesions and were significantly more likely to have required subcutaneous fluid administration. These findings identify aEPEC to be prevalent in kittens and a significant primary or contributing cause of intestinal inflammation, diarrhea, dehydration, and associated mortality in kittens., (Copyright © 2017 American Society for Microbiology.)

Practical relevance: Trichomonosis of the large intestine of the cat was described as a cause of chronic diarrhea over 20 years ago. The trichomonad was identified as Tritrichomonas foetus, with a genotype that is distinct from venereal T foetus of cattle. Clinical challenges: Despite multiple means for diagnosis of the infection, including light microscopy, protozoal culture and PCR amplification using species-specific primers, tests with even greater sensitivity are needed. Feline trichomonosis is resistant to all commonly used antiprotozoal drugs. Ronidazole is currently the only drug demonstrated to be effective in eliminating the infection from cats; however, this drug has a narrow safety margin and clinical resistance is increasingly recognized. The more we learn about trichomonosis in cats, the more complicated and controversial the infection has become, ranging from what we should call the organism to whether we should even bother trying to treat it. Global importance: Feline trichomonosis is recognized to occur worldwide and is regarded as one of the most common infectious causes of colitis in the domestic cat. The infection is widespread in catteries and shelters; and, while remission of diarrhea may occur over time, persistence of the infection is common. Evidence base: This review provides a comprehensive examination of what is currently known about feline trichomonosis and pinpoints areas, based on the authors' opinion, where further research is needed.

Background: Megaesophagus carries a poor to guarded prognosis due to death from aspiration pneumonia. Options for medical management of regurgitation are limited to strategic oral or gastrostomy tube feeding., Objectives: To describe the use and efficacy of intermittent esophageal suctioning to prevent regurgitation and associated episodes of aspiration pneumonia in dogs with megaesophagus., Animals: Four dogs with acquired idiopathic megaesophagus and recurrent aspiration pneumonia., Methods: Retrospective review of medical records of dogs with megaesophagus in which intermittent suctioning of esophageal content was employed for management of recurrent aspiration pneumonia., Results: Intermittent suctioning of the esophagus was initiated in 4 dogs after failure of strict gastrostomy tube feeding failed to prevent regurgitation and repeated episodes of aspiration pneumonia. Suctioning was accomplished by esophagostomy tube in 3 dogs and per os in 1 dog. After initiation of esophageal suctioning, dogs survived for a median of 13.5 additional months (range, 10-30 months) during which time 2 dogs had no additional episodes of aspiration pneumonia and 2 dogs had infrequent episodes of pneumonia, but aspiration was suspected to be a contributing factor in their death. Complications included clogging of the esophagostomy tube, esophagostomy site infections, and esophagitis., Conclusions and Clinical Importance: Use of intermittent esophageal suctioning in dogs with megaesophagus that continue to regurgitate despite gastrostomy tube feedings can reduce or abolish clinical episodes of aspiration pneumonia., (Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Objectives: Coxiella burnetii is an obligate intracellular bacterium that is found worldwide, is associated or suggested to be associated with reproductive abnormalities in a number of species including cats, and is the cause of Q fever in humans. In a previous study, C burnetii DNA was amplified from the uterine tissues of 8.5% of client-owned cats in the USA but reproductive history was unknown and histopathological examination was not performed. In this study, uterine tissues of 26 normal cats and 11 cats with histopathological evidence of uterine disease or other reproductive abnormalities were evaluated for the presence of C burnetii., Methods: A PCR assay that amplifies the repetitive transposon-like region (Trans 1 and 2) and a PCR assay that amplifies the IS-1111-insertion sequence (IS-1111) were optimised and applied to the DNA extracts. The sensitivity threshold of both PCR assays was 12 pg/µl. Positive samples were evaluated for the presence of the organism using immunohistochemistry performed on paraffin-embedded tissue., Results: Amplicons of the expected size developed in three samples (one from a cat with reproductive abnormalities) in the IS-1111 assay; however, there was not enough DNA for sequence analysis. Immunohistochemical analysis was used to further evaluate these three samples and was negative for C burnetii. While C burnetii could not be confirmed by sequence analysis or immunohistochemistry, the PCR positive prevalence rate (8.1%) was similar to that published previously., Conclusions and Relevance: Biosafety precautions should be taken when working with cats that are aborting or parturient. Further research should be performed to evaluate the role that C burnetii may play in reproductive abnormalities in cats., (© ISFM and AAFP 2015.)

Almost 20 years has passed since trichomonosis was first recognized as a potential cause of diarrhea in domestic cats. Despite progress in confirming disease causation, developing means for diagnosis, and identifying approaches to treatment of the infection, we still know very little about how this parasite causes diarrhea. With increasing recognition of resistance of trichomonosis to treatment with 5-nitroimidazole drugs, new treatment strategies based on an understanding of disease pathogenesis are needed. In this review, lessons learned from the pathogenesis of venereal trichomonosis in people and cattle are applied to clinical observations of trichomonosis in cats in effort to generate insight into areas where further research may be beneficial., (Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Cyniclomyces guttulatus, a gastrointestinal yeast of rabbits, is considered an uncommon, nonpathogenic, "pass through" organism and possible opportunistic pathogen in dogs that consume rabbit feces. This retrospective study aimed to characterize the presenting complaint, clinical findings, location of organisms, and final diagnosis of dogs in which yeast morphologically consistent with C. guttulatus were identified at a veterinary teaching hospital from 2006-2013. The prevalence of C. guttulatus infection in a general population of dogs from a regional animal shelter was also determined. Nineteen dogs were retrospectively identified as diagnosed with C. guttulatus infection. Among these, 79% presented with a chief complaint and/or clinical signs consistent with gastrointestinal tract disease. The most common clinical sign was chronic diarrhea. The majority of dogs had C. guttulatus identified cytologically within samples obtained from the gastrointestinal tract; however, four dogs had C. guttulatus identified in non-gastrointestinal tract samples, including a nasal biopsy (one dog) and urine (three dogs). C. guttulatus was not identified in any of 105 shelter dogs evaluated, suggesting low prevalence of C. guttulatus in our region. These findings suggest that additional studies to determine if C. guttulatus is a potential cause or consequence of gastrointestinal illness in dogs may be warranted.

Background: The cause of gallbladder mucocele (GBM) formation in dogs currently is unknown. Many available drugs represent a newer generation of xenobiotics that may predispose dogs to GBM formation., Objective: To determine if there is an association between the histologic diagnosis of GBM in dogs and administration of selected drugs., Animals: Eighty-one dogs with a histologic diagnosis of GBM and 162 breed, age, and admission date-matched control dogs from a single referral institution., Methods: Medical records of dogs with GBM and control dogs from 2001 to 2011 were reviewed. Owner verification of drug history was sought by a standard questionnaire. Reported use of heartworm, flea, and tick preventatives as well as nonsteroidal anti-inflammatory drugs, analgesics, corticosteroids, or medications for treatment of osteoarthritis was recorded., Results: Dogs with GBM were 2.2 times as likely to have had reported use of thyroxine (as a proxy for the diagnosis of hypothyroidism) as control dogs (95% confidence interval [CI], 0.949-5.051), 3.6 times as likely to have had reported treatment for Cushing's disease (95% CI, 1.228-10.612), and 2.3 times as likely to have had reported use of products containing imidacloprid (95% CI, 1.094-4.723). Analysis of a data subset containing only Shetland sheepdogs (23 GBM and 46 control) indicated that Shetland sheepdogs with GBM formation were 9.3 times as likely to have had reported use of imidacloprid as were control Shetland sheepdogs (95% CI, 1.103-78.239)., Conclusions and Clinical Importance: This study provides evidence for an association between selected drug use and GBM formation in dogs. A larger epidemiologic study of Shetland sheepdogs with GBM formation and exposure to imidacloprid is warranted., (Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Mucosal protection of the gallbladder is vital yet we know very little about the mechanisms involved. In domestic dogs, an emergent syndrome referred to as gallbladder mucocele formation is characterized by excessive secretion of abnormal mucus that results in obstruction and rupture of the gallbladder. The cause of gallbladder mucocele formation is unknown. In these first mechanistic studies of this disease, we investigated normal and mucocele-forming dog gallbladders to determine the source, identity, biophysical properties, and protein associates of the culprit mucins with aim to identify causes for abnormal mucus behavior. We established that mucocele formation involves an adoptive excess secretion of gel forming mucins with abnormal properties by the gallbladder epithelium. The mucus is characterized by a disproportionally significant increase in Muc5ac relative to Muc5b, defective mucin un-packaging, and mucin-interacting innate defense proteins that are capable of dramatically altering the physical and functional properties of mucus. These findings provide an explanation for abnormal mucus behavior and based on similarity to mucus observed in the airways of people with cystic fibrosis, suggest that abnormal mechanisms for maintenance of gallbladder epithelial hydration may be an instigating factor for mucocele formation in dogs.

An 8-week-old male domestic shorthair was presented to the Internal Medicine Service at North Carolina State University for regurgitation. Radiographic diagnosis of generalized esophageal dilation and failure of esophageal peristalsis were compatible with diagnosis of congenital megaesophagus. Endoscopic examination of the esophagus revealed a fibrous stricture just orad to the lower esophageal sphincter. Conservative management to increase the body condition and size of the kitten consisted of feeding through a gastrostomy tube, during which time the esophagus regained normal peristaltic function, the stricture orifice widened in size and successful balloon dilatation of the stricture was performed. Esophageal endoscopy should be considered to rule out a stricture near the lower esophageal sphincter in kittens with radiographic findings suggestive of congenital megaesophagus. Management of such kittens by means of gastrostomy tube feeding may be associated with a return of normal esophageal motility and widening of the esophageal stricture, and facilitate subsequent success of interventional dilation of the esophageal stricture., (© ISFM and AAFP 2014.)

A voided urine sample, obtained from a 13-year-old intact male dog residing in a laboratory animal research facility, was observed to contain biflagellate protozoa 5 days following an episode of gross hematuria. The protozoa were identified as belonging to the class Kinetoplastea on the basis of light microscopic observation of Wright-Giemsa-stained urine sediment in which the kinetoplast was observed basal to 2 anterior flagella. A polymerase chain reaction (PCR) assay using primers corresponding with conserved regions within the 18S ribosomal RNA gene of representative kinetoplastid species identified nucleotide sequences with 100% identity to Parabodo caudatus. Parabodo caudatus organisms were unable to be demonstrated cytologically or by means of PCR in samples collected from the dog's environment. The dog had a history of 50 complete urinalyses performed over the 12-year period preceding detection of P. caudatus, and none of these were noted to contain protozoa. Moreover, the gross hematuria that was documented 5 days prior to detection of P. caudatus had never before been observed in this dog. Over the ensuing 2.5 years of the dog's life, 16 additional complete urinalyses were performed, none of which revealed the presence of protozoa. Bodonids are commonly found in soil as well as in freshwater and marine environments. However, P. caudatus, in particular, has a 150-year-long, interesting, and largely unresolved history in people as either an inhabitant or contaminant of urine. This historical conundrum is revisited in the current description of P. caudatus as recovered from the urine of a dog., (© 2014 The Author(s).)

Trichomonads are obligate protozoan parasites most renowned as venereal pathogens of the reproductive tract of humans and cattle. Recently, a trichomonad highly similar to bovine venereal Tritrichomonas foetus but having a unique tropism for the intestinal tract was recognized as a significant cause of colitis in domestic cats. Despite a high prevalence, worldwide distribution, and lack of consistently effective drugs for treatment of the infection, the cellular mechanisms of T. foetus pathogenicity in the intestinal tract have not been examined. The aims of this study were to determine the pathogenic effect of feline T. foetus on porcine intestinal epithelial cells, the dependence of T. foetus pathogenicity on adhesion of T. foetus to the intestinal epithelium, and the identity of mediators responsible for these effects. Using an in vitro coculture approach to model feline T. foetus infection of the intestinal epithelium, these studies demonstrate that T. foetus promotes a direct contact-dependent activation of intestinal epithelial cell apoptosis signaling and progressive monolayer destruction. Moreover, these pathological effects were demonstrated to be largely dependent on T. foetus cell-associated cysteine protease activity. Finally, T. foetus cysteine proteases were identified as enabling cytopathic effects by promoting adhesion of T. foetus to the intestinal epithelium. The present studies are the first to examine the cellular mechanisms of pathogenicity of T. foetus toward the intestinal epithelium and support further investigation of the cysteine proteases as virulence factors in vivo and as potential therapeutic targets for ameliorating the pathological effects of intestinal trichomonosis., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Approximately 15% of foster kittens die before 8 weeks of age, with most of these kittens demonstrating clinical signs or postmortem evidence of enteritis. While a specific cause of enteritis is not determined in most cases, these kittens are often empirically administered probiotics that contain enterococci. The enterococci are members of the commensal intestinal microbiota but also can function as opportunistic pathogens. Given the complicated role of enterococci in health and disease, it would be valuable to better understand what constitutes a "healthy" enterococcal community in these kittens and how this microbiota is impacted by severe illness. In this study, we characterized the ileum mucosa-associated enterococcal community of 50 apparently healthy and 50 terminally ill foster kittens. In healthy kittens, Enterococcus hirae was the most common species of ileum mucosa-associated enterococci and was often observed to adhere extensively to the small intestinal epithelium. These E. hirae isolates generally lacked virulence traits. In contrast, non-E. hirae enterococci, notably Enterococcus faecalis, were more commonly isolated from the ileum mucosa of kittens with terminal illness. Isolates of E. faecalis had numerous virulence traits and multiple antimicrobial resistances. Moreover, the attachment of Escherichia coli to the intestinal epithelium was significantly associated with terminal illness and was not observed in any kitten with adherent E. hirae. These findings identify a significant difference in the species of enterococci cultured from the ileum mucosa of kittens with terminal illness compared to the species cultured from healthy kittens. In contrast to prior case studies that associated enteroadherent E. hirae with diarrhea in young animals, these controlled studies identified E. hirae as more often isolated from healthy kittens and adherence of E. hirae as more common and extensive in healthy kittens than in sick kittens.

Ronidazole (RDZ) is the only known effective treatment for feline diarrhea caused by Tritrichomonas foetus. This study aimed to develop guar gum-coated colon-targeted tablets of RDZ and to determine the pharmacokinetics of this delayed-release formulation in cats. Guar gum-coated tablets were administered orally once to five healthy cats (mean dose 32.3 mg/kg). The tablets were then administered once daily for 5 days to four cats (mean dose 34.5 mg/kg), and absorption studies repeated on day 5. Plasma was collected and analyzed for RDZ concentration, and pharmacokinetic noncompartmental and deconvolution analysis were performed on the data. There was negligible RDZ release until after 6 h, and a delayed peak plasma concentration (mean Cmax 28.9 μg/mL) at approximately 14.5 h, which coincides with colonic arrival in cats. Maximum input rate (mg/kg per hour) occurred between 6 and 16 h. This delayed release of ronidazole from guar gum-coated tablets indicates that release of RDZ may be delayed to deliver the medication to a targeted area of the intestine. Repeated dosing with guar gum tablets to steady-state did not inhibit drug bioavailability or alter the pharmacokinetics. Such targeted RDZ drug delivery may provide improved efficacy and reduce adverse effects in cats., (© 2012 John Wiley & Sons Ltd.)

Tritrichomonas foetus (TF) is a protozoan that infects the feline ileum and colon resulting in chronic diarrhea. Up to 30% of young purebred cats are infected with TF and the infection is recognized as pandemic. Only a single drug, characterized by a narrow margin of safety and emerging development of resistance, is effective for treatment. While the venereal pathogenicity of bovine TF is attributed to adherence to uterovaginal epithelium, the pathogenesis of diarrhea in feline TF infection is unknown. The aim of this study was to establish an in vitro model of feline TF adhesion to intestinal epithelium. Confluent monolayers of porcine intestinal epithelial cells (IPEC-J2) were infected with axenic cultures of feline TF that were labeled with [(3)H] thymidine or CFSE and harvested at log-phase. The effect of multiplicity and duration of infection, viability of TF, binding competition, formalin fixation and cytoskeletal inhibitors on adherence of feline TF to IPEC-J2 monolayers was quantified by liquid scintillation counting and immunofluorescence. [(3)H] thymidine and CFSE-labeled TF reproducibly adhered to IPEC-J2 monolayers. Clinical isolates of feline TF adhered to the intestinal epithelium in significantly greater numbers than Pentatrichomonas hominis, the latter of which is a presumably nonpathogenic trichomonad. Adhesion of TF required viable trophozoites but was independent of cytoskeletal activity. Based on saturation and competition binding experiments, adherence of feline TF to the epithelium occurred via specific receptor-ligand interactions. The developed model provides a valuable resource for assessing pathogenic mechanisms of feline TF and developing novel pharmacologic therapies for blocking the adhesion of feline TF to the intestinal epithelium., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Canine granulomatous hepatitis is an uncommon morphologic diagnosis that has been associated with a variety of diseases, including a number of systemic infectious etiologies. Formalin-fixed, paraffin-embedded (FFPE) tissues are typically the only source of liver tissue remaining for additional testing for the presence of infectious disease within granulomas. It is unclear if the more common infectious culprits of granulomatous hepatitis can be identified from such specimens. The aim of the current study was to retrospectively investigate archival FFPE liver tissue from dogs with granulomatous hepatitis for the presence of infectious agents. Semiquantitative analysis of copper accumulation in liver specimens was also performed. Medical records were examined for recorded evidence of systemic infectious disease diagnosis. Formalin-fixed, paraffin-embedded liver was prospectively evaluated for infectious agents via differential staining techniques (n = 13), eubacterial fluorescent in situ hybridization (n = 11), and Bartonella polymerase chain reaction assays (n = 15). An infectious cause of granulomatous hepatitis was not identified within liver tissue from any dog using these diagnostic methodologies. Six out of 25 (24%) dogs were diagnosed with concurrent systemic or localized bacterial infections at the time of presentation. Nine out of 17 (53%) dogs had excessive hepatic copper accumulation when evaluated by a semiquantitative histologic grading scheme or quantitative copper analysis. As definitive infectious causes of granulomatous hepatitis were not identified within archival liver biopsy samples, it was concluded that investigation of infectious etiologies within FFPE liver specimens using these diagnostic approaches may be of low yield.

Background & Aims: Although we are beginning to understand where, when, and how intestinal epithelial cells are shed, physiologically, less is understood about alterations in cell fate during minimally invasive epithelial infections. We used a piglet model of Cryptosporidium parvum infection to determine how elimination of infected enterocytes is balanced with the need to maintain barrier function., Methods: We studied the effects of enterocyte shedding by C parvum-infected ileum on barrier function ex vivo with Ussing chambers. The locations and activities of caspase-3, nuclear factor κB (NF-κB), and inhibitor of apoptosis proteins (IAP) were assayed by enzyme-linked immunosorbent assay, immunoblot, and tissue immunoreactivity analyses and using specific pharmacologic inhibitors. The location, specificity, and magnitude of enterocyte shedding were quantified using special stains and light microscopy., Results: Infection with C parvum activated apoptotic signaling pathways in enterocytes that resulted in cleavage of caspase-3. Despite caspase-3 cleavage, enterocyte shedding was confined to villus tips, coincident with apoptosis, and observed more frequently in infected cells. Epithelial expression of X-linked inhibitor of apoptosis protein (XIAP), activation of NF-κB, and proteasome activity were required for control of cell shedding and barrier function. The proteasome blocked activity of caspase-3; this process was mediated by expression of XIAP, which bound to cleaved caspase-3., Conclusions: We have identified a pathway by which villus epithelial cells are maintained during C parvum infection. Loss of barrier function is reduced by active retention of infected enterocytes until they reach the villus tip. These findings might be used to promote clearance of minimally invasive enteropathogens, such as by increasing the rate of migration of epithelial cells from the crypt to the villus tip., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The urinary bladder is a common site of bacterial infection with a majority of cases attributed to uropathogenic Escherichia coli. Sequelae of urinary tract infections (UTIs) include the loss of urothelial barrier function and subsequent clinical morbidity secondary to the permeation of urine potassium, urea and ammonia into the subepithelium. To date there has been limited research describing the mechanism by which this urothelial permeability defect develops. The present study models acute uropathogenic E. coli infection in vitro using intact canine bladder mucosa mounted in Ussing chambers to determine whether infection induces primarily a transcellular or paracellular permeability defect. The Ussing chamber sustains tissue viability while physically separating submucosal and lumen influences, so this model is ideal for quantitative measurement of transepithelial electrical resistance (TER) to assess alterations of urothelial barrier function. Using this model, changes in both tissue ultrastructure and TER indicated that uropathogenic E. coli infection promotes a paracellular permeability defect associated with the failure of umbrella cell tight junction formation and umbrella cell sloughing. In addition, bacterial interaction with the urothelium promoted secretion of cytokines from the urinary bladder with bioactivity capable of modulating epithelial barrier function including tumour necrosis factor-α, interleukin (IL)-6 and IL-15. IL-15 secretion by the infected bladder mucosa is a novel finding and, because IL-15 plays key roles in reconstitution of tight junction function in damaged intestine, this study points to a potential role for IL-15 in UTI-induced urothelial injury., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Trichomonads have been infrequently reported in the feces of dogs where their pathogenicity remains uncertain. It is currently unknown whether Tritrichomonas foetus or Pentatrichomonas hominis is identified more commonly in dogs with trichomonosis or how often these infections are accompanied by concurrent enteric infectious agents. The objective of this study was to determine the identity of trichomonads present in a series of 38 unsolicited canine diarrheic fecal samples submitted for T. foetus diagnostic polymerase chain reaction (PCR) testing between 2007 and 2010. We also examined each fecal sample for an association of trichomonosis with concurrent infection using a convenient real-time PCR panel for nine gastrointestinal pathogens. P. hominis, T. foetus, or both were identified by PCR in feces of 17, 1, and 1 dogs respectively. Feces from the remaining 19 dogs were PCR negative for T. foetus, P. hominis and using broader-spectrum Trichomonadida primers. The total number and specific identities of concurrent enteropathogens identified did not differ between fecal samples from dogs that were or were not identified by PCR as infected with trichomonads. These results suggest that P. hominis infection is more frequently identified than T. foetus infection in diarrheic dogs with trichomonosis and that concurrent enteropathogen infection is common in this population., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Investigation of bacteria involved in pathogenesis of necrotizing enterocolitis (NEC) is limited by infant fragility, analysis restricted to feces, use of culture-based methods, and lack of clinically-relevant animal models. This study used a unique preterm piglet model to characterize spontaneous differences in microbiome composition of NEC-predisposed regions of gut.  Preterm piglets (n=23) were cesarean-delivered and nurtured for 30 hours over which time 52% developed NEC. Bacterial DNA from ileal content, ileal mucosa, and colonic mucosa were PCR amplified, subjected to terminal restriction fragment length polymorphism (TRFLP) analysis and targeted 16S rDNA qPCR.  Preterm ileal mucosa was specifically bereft in diversity of bacteria compared to ileal content and colonic mucosa. Preterm ileum was restricted to representation by only Proteobacteria, Firmicutes, Cyanobacteria and Chloroflexi. In piglets with NEC, ileal mucosa was uniquely characterized by increases in number of Firmicutes and diversity of phyla to include Actinobacteria and uncultured bacteria. Five specific TRFLP profiles, corresponding in closest identity to Clostridium butyricum, C. neonatale, C. proteolyticum, Streptomyces spp., and Leptolyngbya spp., were significantly more prevalent or observed only among samples from piglets with NEC. Total numbers of Clostridium spp. and C. butyricum were significantly greater in samples of NEC ileal mucosa but not ileal content or colonic mucosa. These results provide strong support for ileal mucosa as a focus for investigation of specific dysbiosis associated with NEC and suggest a significant role for Clostridium spp., and members of the Actinobacteria and Cyanobacteria in the pathogenesis of NEC in preterm piglets.

Ronidazole (RDZ) is an effective treatment for feline Tritrichomonas foetus infection, but has produced neurotoxicity in some cats. An understanding of the disposition of RDZ in cats is needed in order to make precise dosing recommendations. Single-dose pharmacokinetics of intravenous (IV) RDZ and immediate-release RDZ capsules were evaluated. A single dose of IV RDZ (mean 9.2mg/kg) and a 95mg immediate-release RDZ capsule (mean 28.2mg/kg) were administered to six healthy cats in a randomized crossover design. Plasma samples were collected for 48 h and assayed for RDZ using high pressure liquid chromatography (HPLC). Systemic absorption of oral RDZ was rapid and complete, with detection in the plasma of all cats by 10 min after dosing and a bioavailability of 99.64 (±16.54)%. The clearance of RDZ following IV administration was 0.82 (±0.07) ml/kg/min. The terminal half-life was 9.80 (±0.35) and 10.50 (±0.82) h after IV and oral administration, respectively, with drug detectable in all cats 48h after both administrations. The high oral bioavailability of RDZ and slow elimination may predispose cats to neurotoxicity with twice-daily administration. Less frequent administration should be considered for further study of effective treatment of T foetus-infected cats., (Copyright © 2011 ISFM and AAFP. Published by Elsevier Ltd. All rights reserved.)

During early urinary tract infection (UTI) the interplay between invading bacteria and the urothelium elicits a mucosal response aimed at clearing infection. Unfortunately, the resultant inflammation and associated local tissue injury are responsible for patient symptoms. Interleukin-6 (IL-6), a cytokine released during acute UTI, has both pro- and anti-inflammatory effects on other body systems. Within the urothelium, the IL-6 native-tissue origin, the target cell type(s), and ultimate effect of the cytokine on target cells are largely unknown. In the present study we modeled the UTI IL-6 response ex vivo using canine bladder mucosa mounted in Ussing chambers to determine the inflammatory and reparative role of IL-6. We demonstrated that uropathogenic Escherichia coli infection stimulates the synthesis of IL-6 by all urothelial cell layers, with the urothelial cells alone representing the only site of unequivocal IL-6 receptor expression. Autocrine effects of IL-6 were supported by the activation of urothelial STAT3 signaling and SOCS3 expression. Using exogenous IL-6, a microarray approach, and quantitative reverse transcriptase PCR (q-RT-PCR), 5 target genes (tumor necrosis factor alpha, interleukin-1β, matrix metallopeptidase 2, heparan sulfate d-glucosaminyl 3-O-sulfotransferase 3A1, and hyaluronan synthase 2) that have direct or indirect roles in promoting a proinflammatory state were identified. Two of these genes, heparan sulfate d-glucosaminyl 3-O-sulfotransferase 3A1 and hyaluronan synthase 2, are also potentially important mediators of wound repair via the production of glycosaminoglycan components. These findings suggest that IL-6 secretion during acute UTI may serve a dual biological role by initiating the inflammatory response while also repairing urothelial defenses.

In the present study a highly species-specific oligonucleotide sequence of Tritrichomonas foetus 18S rRNA was used to design an antisense probe for identification of T. foetus in formalin-fixed and paraffin-embedded histological specimens by means of fluorescence in situ hybridization (FISH). Using archival histological specimens from several species with light microscopic evidence of intestinal trichomoniasis, and under optimized hybridization conditions, the probe positively identified trichomonads in colonic specimens from piglets and a kitten with PCR-confirmed T. foetus infection. Neither positive hybridization of the probe or PCR amplification of T. foetus DNA was observed in histological specimens from hamster (Tritrichomonas muris), turkey, nor mouse (Entamoeba muris) intestinal protozoal infections. Sequence-specific binding of the probe was further verified by successfully out-competing the hybridization with 10 x molar excess unlabeled probe and failure of a labeled sense probe to hybridize. The FISH assay described here enables simultaneous location and molecular identification of T. foetus in formalin-fixed and paraffin-embedded histological specimens of intestinal trichomoniasis. The methods employed are likely to also be applicable to probes designed for specific recognition of other trichomonad species, especially in mammalian tissue where red blood cell auto-fluorescence can be easily differentiated from the hybridization signal of trichomonads., ((c) 2010 Elsevier B.V. All rights reserved.)

The bacterial causes of diarrhea can be frustrating to identify, and it is likely that many remain undiagnosed. The pathogenic potential of certain bacteria becomes less ambiguous when they are observed to intimately associate with intestinal epithelial cells. In the present study we sought to retrospectively characterize the clinical, in situ molecular, and histopathological features of enteroadherent bacteria in seven unrelated kittens that were presumptively diagnosed with enteropathogenic Escherichia coli (EPEC) on the basis of postmortem light microscopic and, in some cases, microbiological examination. Characterization of the enteroadherent bacteria in each case was performed by Gram staining, in situ hybridization using fluorescence-labeled oligonucleotide probes, PCR amplification of species-specific gene sequences, and ultrastructural imaging applied to formalin-fixed paraffin-embedded sections of intestinal tissue. In only two kittens was EPEC infection confirmed. In the remaining five kittens, enteroadherent bacteria were identified as Enterococcus spp. The enterococci were further identified as Enterococcus hirae on the basis of PCR amplification of DNA extracted from the formalin-fixed, paraffin-embedded tissue and amplified by using species-specific primers. Transmission electron microscopy of representative lesions from E. coli- and Enterococcus spp.-infected kittens revealed coccobacilli adherent to intestinal epithelial cells without effacement of microvilli or cup-and-pedestal formation. Enterococci were not observed, nor were DNA sequences amplified from intestinal tissue obtained from age-matched kittens euthanized for reasons unrelated to intestinal disease. These studies suggest that E. hirae may be a common cause of enteroadherent bacterial infection in pre-weaning-age kittens and should be considered in the differential diagnosis of bacterial disease in this population.

Background: The mainstays of treatment for clinically important trichomonad infections are the 5-nitroimidazoles. Metronidazole resistance of feline Tritrichomonas foetus is presumed because of common treatment failure, and tinidazole does not consistently eradicate infection. To date, ronidazole is the only drug demonstrated as effective for treatment of cats infected with T. foetus., Objective: To document in vivo treatment failure and identify underlying causes and in vitro conditions of resistance of feline T. foetus to ronidazole., Animals: Two intact male Abyssinians failing>or=5 courses of treatment with increasing doses of 5-nitroimidazole drugs. An intact male Abyssinian documented to clear infection after treatment with a single course of ronidazole., Methods: T. foetus isolates were cultured from feces and tested in vitro for susceptibility to ronidazole under aerobic and anaerobic culture conditions. A urogenital nidus of T. foetus infection was investigated by culture, polymerase chain reaction, or immunohistochemical testing of urogenital specimens., Results: Resistance to ronidazole under aerobic conditions was uniquely identified in T. foetus isolated from cats with well-documented treatment failure. Treatment failure could not be attributed to reinfection, inappropriate treatment protocol, or presence of a urogenital nidus of infection., Conclusions and Clinical Importance: Clinical resistance to metronidazole, low efficacy of tinidazole, and present documentation of in vivo and in vitro resistance to ronidazole in some cats are consistent with a high level of cross resistance of feline T. foetus to 5-nitroimidazole drugs. Current lack of alternative drugs with clinical efficacy against feline T. foetus suggests that active investigation of other treatment approaches is warranted.

The regulation of the glomerular filtration rate (GFR) is a particularly important and challenging concept for students to integrate into a memorable framework for building further knowledge and solving clinical problems. In this study, 76 first-year veterinary students and 19 veterinarians in clinical specialty training (house officers) participated in separate online exercises to evaluate the use of a computer-animated model of GFR regulation (www.aamc.org/mededportal) on learning outcome. Students were randomly allocated to study either the animated model or written materials before completion of a 10-question multiple-choice quiz. House officers completed a 35-question test before and after study of the animated model. Both groups completed a survey about the learning exercise. The ability of the model to enhance learning was demonstrated by a significant improvement (P < 0.001) in the test performance of house officers after studying the model. The model performed similarly to written materials alone in affecting the subsequent quiz performance of the students. The majority of students and house officers agreed or strongly agreed that the animated model was easy to understand, improved their knowledge and appreciation of the importance of GFR regulation, and that they would recommend the model to peers. Most students [63 of 76 students (83%)] responded that they would prefer the use of the animated model alone over the study of written materials but acknowledged that a combination of hardcopy written notes and the animated model would be ideal. A greater applicability of the model to more advanced students and an introduction in a didactic setting before individual study were suggested by the house officers. The results of this study suggest that the animated model is a useful, effective, and well-received tool for learning and creating a visual memory of the regulatory mechanisms of GFR.

We hypothesized that unrecognized differences in epithelial expression of inducible nitric oxide synthase (iNOS), resulting from engineered immunodeficiency, could explain the contradictory findings of prior studies regarding the importance of nitric oxide (NO) in murine models of Cryptosporidium parvum infection. Severe combined immunodeficient mice (SCID) failed to constitutively or inducibly express epithelial iNOS or increase NO synthesis in response to C. parvum infection. In contrast, mice lacking IFN-gamma alone induced both epithelial iNOS expression and NO synthesis in response to infection. Accordingly, lymphocytes mediate epithelial expression of iNOS and NO synthesis independent of IFN-gamma in response to C. parvum infection. These findings in large part explain the contradictory conclusions of prior studies regarding the role of iNOS in C. parvum infection.

Objective: To determine whether Tritrichomonas foetus infection resides in reproductive tract tissues from cats housed for breeding and for which a high prevalence of colonic T foetus infection has been reported., Animals: 61 purebred cats in 36 catteries undergoing elective ovariohysterectomy or castration and for which reproductive tract tissues, feces, and a reproductive history were obtained., Procedures: Reproductive tract tissues were examined for T foetus via light microscopy, immunohistochemical analysis, and PCR assay. History of reproductive tract disease was examined to detect statistical associations with identified or reported exposure to colonic T foetus infection., Results: 15 of 61 (25%) cats and 22 of 33 (67%) catteries were identified with active or reported T foetus infection. Light microscopic, immunohistochemical, or molecular evidence of T foetus infection of the reproductive tract was not detected in any cats, including 15 cats with colonic T foetus infection, 29 cats residing in a cattery in which T foetus-infected cats were identified, and 8 cats for which gross or light microscopic evidence of reproductive tract disease was identified. There were no differences in total number of litters, number of litters per breeding, kitten mortality rate, or birth defects between cats or catteries infected with T foetus and those for which T foetus infection was not identified., Conclusions and Clinical Relevance: No evidence of reproductive tract colonization by T foetus was detected in this study. Accordingly, it is unlikely that reproductive tract infection with T foetus plays an important role in overall disease transmission.

Understanding reticulorumen motility is important to the assessment of ruminant health and optimal production, and in the recognition, diagnosis, and treatment of disease. Accordingly, the teaching of reticulorumen motility is a staple of all veterinary curricula. This teaching has historically been based on written descriptions, line drawings, or pressure tracings obtained during contraction sequences. We developed an animated model of reticulorumen motility and hypothesized that veterinary students would prefer use of the model over traditional instructional methods. First-year veterinary students were randomly allocated to one of two online learning exercises: with the animated model (Group A) or with text and line drawings (Group B) depicting reticulorumen motility. Learning was assessed with a multiple-choice quiz and feedback on the learning alternatives was obtained by survey. Seventy-four students participated in the study, including 38/42 in Group A and 36/36 in Group B. Sixty-four out of 72 students (89%) responded that they would prefer use of the animated model if only one of the two learning methods was available. A majority of students agreed or strongly agreed that the animated model was easy to understand and improved their knowledge and appreciation of the importance of reticulorumen motility, and would recommend the model to other veterinary students. Interestingly, students in Group B achieved higher scores on examination than students in Group A. This could be speculatively attributed to the inclusion of an itemized list of contraction sequences in the text provided to Group B and failure of Group A students to read the text associated with the animations.