Acquired dysfunction of CFTR underlies cystic fibrosis-like disease of the canine gallbladder.

Mucocele formation in dogs is a unique and enigmatic muco-obstructive disease of the gallbladder caused by amassment of abnormal mucus that bears striking pathological similarity to cystic fibrosis. We investigated the role of CFTR in the pathogenesis of this disease. The location and frequency of disease-associated variants in the coding region of CFTR was 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 was quantified in control and mucocele gallbladders by NanoString, Western blotting, immunofluorescence imaging, and studies in Ussing chambers. Our results establish 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 to 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 was accompanied by decreases in mRNA for the Cl-/HCO3- 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 underlying failure of CFTR function.

Gallbladder microbiota in healthy dogs and dogs with mucocele formation.

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.

Randomized placebo-controlled trial of feline-origin Enterococcus hirae probiotic effects on preventative health and fecal microbiota composition of fostered shelter kittens.

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.

Dysbiosis of fecal microbiota in cats with naturally occurring and experimentally induced Tritrichomonas foetus infection.

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.

Comparative Genomics of Atypical Enteropathogenic Escherichia coli from Kittens and Children Identifies Bacterial Factors Associated with Virulence in Kittens.

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.

Association of fecal sample collection technique and treatment history with Tritrichomonas foetus polymerase chain reaction test results in 1717 cats.

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.

Influence of the intestinal microbiota on disease susceptibility in kittens with experimentally-induced carriage of atypical enteropathogenic Escherichia coli.

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.

Mortality in kittens is associated with a shift in ileum mucosa-associated enterococci from Enterococcus hirae to biofilm-forming Enterococcus faecalis and adherent Escherichia coli.

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.

Proteasome inhibition of pathologic shedding of enterocytes to defend barrier function requires X-linked inhibitor of apoptosis protein and nuclear factor κB.

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.

Acute necrotizing enterocolitis of preterm piglets is characterized by dysbiosis of ileal mucosa-associated bacteria.

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.

Induction of arginase II by intestinal epithelium promotes the uptake of L-arginine from the lumen of Cryptosporidium parvum-infected porcine ileum.

OBJECTIVES: To determine the specific transport system activities and expression of transporter genes responsible for uptake of L-arginine from the lumen of normal and Cryptosporidium parvum-infected neonatal porcine ileum and the influence of L-arginine catabolic pathways on L-arginine uptake. METHODS: Intact sheets of ileal mucosa from control and C parvum-infected neonatal piglets were mounted in Ussing chambers and the uptake of 14C-L-arginine was determined under initial rate conditions and in the presence of transport system-selective inhibitors. Epithelial expression of L-arginine transporter genes was quantified by real-time reverse transcription polymerase chain reaction. L-Arginine catabolic enzyme expression was examined by immunoblotting epithelial lysates for arginase I and II. The role of intracellular catabolism in promoting the uptake of L-arginine was determined by pharmacological inhibition of nitric oxide synthase and arginase activities. RESULTS: C parvum-infected ileum transported L-arginine at rates equivalent to uninfected epithelium despite profound villous atrophy. This was attributed to enhanced uptake of L-arginine by individual epithelial cells in the infection. There were no differences in L-arginine transport system activities (y(+) and B(0, +)) or level of transporter gene expression (CAT-1, CAT-2A, and ATB(0, +)) between uninfected and C parvum-infected epithelial cells. However, infected epithelia had induced expression of the L-arginine hydrolytic enzyme arginase II and lower concentrations of L-arginine. Furthermore, transport of L-arginine by the infected epithelium was significantly inhibited by pharmacological blockade of arginase. CONCLUSIONS: Intracellular catabolism by arginase II, the induction of which has not been described previously for intestinal epithelium, facilitates uptake of L-arginine by infected epithelium using transport systems that do not differ from those of uninfected cells. Induction of arginase II may limit nitric oxide synthesis by competing with nitric oxide synthase for utilization of L-arginine or promote use of L-arginine for the synthesis of reparative polyamines.

Evaluation of four DNA extraction methods for the detection of Tritrichomonas foetus in feline stool specimens by polymerase chain reaction.

Feces are increasingly valued as practical samples for molecular diagnosis of infectious disease. However, extraction of polymerase chain reaction (PCR) quality DNA from fecal samples can be challenging because of coextraction of PCR inhibitors. Because the type and quantity of PCR inhibitors is influenced by diet, endogenous flora, and concurrent disease, it is unlikely that extraction method performance with human feces can be directly extrapolated to that of domestic cats. In the present study, 4 commercially available DNA extraction methods were examined for their influence on the sensitivity of PCR for the detection of Tritrichomonas foetus in feline stool. DNA was extracted from serially diluted feline-origin T. foetus trophozoites in the absence or presence of feline feces. The ZR Fecal DNA kit was identified as affording the greatest analytical sensitivity and reproducibility and was able to detect >or=10 T. foetus organisms per 100 mg feces in 100% of PCR reactions. Further, the identified extraction method could be completed in the shortest time of all kits tested.

Oral delivery of L-arginine stimulates prostaglandin-dependent secretory diarrhea in Cryptosporidium parvum-infected neonatal piglets.

OBJECTIVES: To determine if oral supplementation with L-arginine could augment nitric oxide (NO) synthesis and promote epithelial defense in neonatal piglets infected with Cryptosporidium parvum. MATERIALS AND METHODS: Neonatal piglets were fed a liquid milk replacer and on day 3 of age infected or not with 10(8) C. parvum oocysts and the milk replacer supplemented with L-arginine or L-alanine. Milk consumption, body weight, fecal consistency, and oocyst excretion were recorded daily. On day 3 postinfection, piglets were euthanized and serum concentration of NO metabolites and histological severity of villous atrophy and epithelial infection were quantified. Sheets of ileal mucosa were mounted in Ussing chambers for measurement of barrier function (transepithelial resistance and permeability) and short-circuit current (an indirect measurement of Cl secretion in this tissue). RESULTS: C. parvum-infected piglets had large numbers of epithelial parasites, villous atrophy, decreased barrier function, severe watery diarrhea, and failure to gain weight. L-Arginine promoted synthesis of NO by infected piglets, which was unaccompanied by improvement in severity of infection but rather promoted epithelial chloride secretion and diarrhea. Epithelial secretion by infected mucosa from L-arginine-supplemented piglets was fully inhibited by the cyclooxygenase inhibitor indomethacin, indicating that prostaglandin synthesis was responsible for this effect. CONCLUSIONS: Results of these studies demonstrate that provision of additional NO substrate in the form of L-arginine incites prostaglandin-dependent secretory diarrhea and does not promote epithelial defense or barrier function of C. parvum-infected neonatal ileum.

Efficacy of tinidazole for treatment of cats experimentally infected with Tritrichomonas foetus.

OBJECTIVE: To determine the efficacy of tinidazole for treatment of cats with experimentally induced Tritrichomonas foetus infection. ANIMALS: 8 specific-pathogen-free kittens. PROCEDURES: Tinidazole was tested for activity against a feline isolate of T foetus in vitro. Kittens were infected orogastrically with the same isolate and treated or not with tinidazole (30 mg/kg, PO, q 24 h for 14 days). Amoxicillin was administered 28 weeks after completion of tinidazole administration to induce diarrhea. Feces were repeatedly tested for T foetus by use of PCR assay and microbial culture for 33 weeks. RESULTS: Tinidazole killed T foetus at concentrations >or= 10 microg/mL in vitro. In experimentally induced infection, tinidazole administered at 30 mg/kg decreased T foetus below the limit of molecular detection in 2 of 4 cats. Recrudescent shedding of T foetus, as elicited by amoxicillin-induced diarrhea, was diminished in cats that received prior treatment with tinidazole. CONCLUSIONS AND CLINICAL RELEVANCE: Although tinidazole decreased the detection of T foetus and treated cats were resistant to later efforts to incite the infection, inability of tinidazole to eradicate infection in many cats poses a serious impediment to the drug's effectiveness in practice.

Optimization of a species-specific polymerase chain reaction assay for identification of Pentatrichomonas hominis in canine fecal specimens.

OBJECTIVE: To determine the optimum reaction conditions and detection limits of PCR assay for identification of Pentatrichomonas hominis in DNA extracted from canine feces. SAMPLE POPULATION: DNA extracted from feces of 4 dogs with diarrhea from which trichomonads were observed, 81 dogs that had feces submitted to a diagnostic laboratory, and 19 dogs residing in a laboratory animal facility. PROCEDURES: Optimum reaction conditions and absolute and practical detection limits of 2 P hominis 18S species-specific primer pairs were determined by use of an in vitro cultivated canine isolate of P hominis in the presence and absence of canine feces. The optimized PCR assay was applied to amplification of P hominis 18S rRNA genes from DNA extracted from the feces of dogs. RESULTS: Under optimized conditions, a primer pair was identified as able to detect as few as 1 P hominis organism/180-mg fecal sample. The PCR assay identified P hominis in diarrheic feces of 4 dogs in which trichomonads were seen by light microscopy. The P hominis genes were not amplified from other fecal samples examined. CONCLUSIONS AND CLINICAL RELEVANCE: Molecular identification of P hominis in feces of 4 dogs with trichomonosis and diarrhea reported here validates the identity of this species in such infections. Sensitive and specific PCR amplification of P hominis 18S rRNA genes from DNA extracted from feces will directly facilitate studies examining pathogenicity of this trichomonad and enable differentiation of P hominis from other known or novel species of trichomonads that may infect the gastrointestinal tract of dogs.

Identification of Pentatrichomonas hominis in feline fecal samples by polymerase chain reaction assay.

Pentatrichomonas hominis is considered to be a commensal protozoan of the vertebrate digestive tract. On the basis of light microscopic examination of feces, some investigators presumptively identified P. hominis as a causative agent of feline diarrhea. However, molecular identification of P. hominis infection in the cat has not been reported. Another trichomonad, Tritrichomonas foetus, is recognized as an intestinal pathogen in cats and often presumptively diagnosed on the basis of the presence of trichomonads in diarrheic feces. It is of importance to determine if cats are natural hosts for P. hominis, as the presence of this organism could result in inaccurate assumption of T. foetus infection. In this study, we used a species-specific PCR assay to identify P. hominis 18S rRNA genes in fecal samples collected from a convenience population of cats in which a high prevalence of T. foetus infection had been previously identified (cat show) or suspected (submitted for T. foetus diagnostic testing). The prevalence of T. foetus infection in these samples was 31% and 28.6%, respectively. P. hominis infection was identified by PCR of DNA extracted from feces of five cats (1.9% and 2.1% of fecal samples, respectively). All cats in which P. hominis was identified were also infected with T. foetus. PCR identification of P. hominis infection in the cat should facilitate future studies to determine the pathogenicity of this species and enable differentiation of P. hominis from other known or as-yet unidentified species of trichomonads that may infect cats.

Neutrophils do not mediate the pathophysiological sequelae of Cryptosporidium parvum infection in neonatal piglets.

Cryptosporidium parvum is a minimally invasive protozoal pathogen of intestinal epithelium that results in villus atrophy, mucosal lipid peroxidation, diarrhea, and diminished barrier function. Influx of neutrophils is a consistent feature of human and animal cryptosporidiosis, and yet their contribution to the pathological sequelae of infection has not been investigated. Accordingly, we used an established neonatal piglet model of C. parvum infection to examine the role of neutrophils in disease pathogenesis by inhibiting their recruitment and activation in vivo using a monoclonal anti-CD18 antibody. Infected piglets were treated daily with anti-CD18 or isotype control immunoglobulin G and euthanized at peak infection, at which time neutrophil infiltrates, lipid peroxidation, severity of infection, and intestinal barrier function were quantified. C. parvum infection resulted in a significant increase in mucosal neutrophil myeloperoxidase activity that was prevented by treatment of piglets with anti-CD18 antibody. Neutrophil recruitment was dependent on mucosal superoxide formation (prevented by treatment of infected piglets with superoxide dismutase). Neutrophils did not contribute to peroxynitrite formation or peroxidative injury of C. parvum-infected mucosa and had no impact on the severity of epithelial infection, villus atrophy, or diarrhea. The presence of neutrophils in C. parvum-infected mucosa was associated with enhanced barrier function that could not be attributed to mucosal elaboration of prostaglandins or stimulation of their synthesis. These studies are the first to demonstrate that neutrophilic inflammation arising in response to infection by a noninvasive epithelial pathogen results in physiologic rather than pathological effects in vivo.

Efficacy of ronidazole for treatment of feline Tritrichomonas foetus infection.

OBJECTIVES: To determine the efficacy of ronidazole (RDZ), tinidazole (TDZ), and metronidazole (MDZ) against Tritrichomonas foetus in vitro and of RDZ for treatment of feline naturally occurring or experimentally induced T. foetus infection. ANIMALS: A cat naturally infected with T. foetus infection and diarrhea. Ten specific-pathogen-free (SPF) kittens. PROCEDURE: RDZ, TDZ, and MDZ were tested for activity against 3 different feline isolates of T. foetus in vitro. RDZ then was administered to a naturally infected cat at 10 mg/kg PO q24h for 10 days. SPF kittens were infected orogastrically with feline T. foetus and treated with either placebo or RDZ (10 mg/kg PO q12h for 14 days). Cats with relapsing infection or those receiving placebo were treated subsequently with RDZ (either 30 or 50 mg/kg PO q12h for 14 days). Feces were examined for T. foetus by direct microscopy, culture, and polymerase chain reaction (PCR) testing weekly. RESULTS: Both RDZ and TDZ killed T. foetus at concentrations >0.1 microg/mL in vitro. In the naturally infected cat, RDZ abolished diarrhea and T. foetus infection for 85 days after treatment, at which time infection and diarrhea relapsed. Retreatment with RDZ eradicated diarrhea and T. foetus infection for over 407 days. In experimentally induced infection, RDZ at 10 mg/kg caused initial improvement, but infection relapsed in all 5 cats 2 to 20 weeks after treatment. At 30 or 50 mg/kg, 10/10 cats were negative for T. foetus infection for follow-up durations of 21 to 30 weeks after treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of RDZ at 30 to 50 mg/kg q12h for 14 days resolved diarrhea and eradicated infection (on the basis of polymerase chain reaction [PCR] testing) in 1 naturally infected cat and 10 experimentally inoculated cats receiving a different isolate of T. foetus.

NF-kappaB-mediated expression of iNOS promotes epithelial defense against infection by Cryptosporidium parvum in neonatal piglets.

Cryptosporidium sp. parasitizes intestinal epithelium, resulting in enterocyte loss, villous atrophy, and malabsorptive diarrhea. We have shown that mucosal expression of inducible nitric oxide (NO) synthase (iNOS) is increased in infected piglets and that inhibition of iNOS in vitro has no short-term effect on barrier function. NO exerts inhibitory effects on a variety of pathogens; nevertheless, the specific sites of iNOS expression, pathways of iNOS induction, and mechanism of NO action in cryptosporidiosis remain unclear. Using an in vivo model of Cryptosporidium parvum infection, we have examined the location, mechanism of induction, specificity, and consequence of iNOS expression in neonatal piglets. In acute C. parvum infection, iNOS expression predominated in the villous epithelium, was NF-kappaB dependent, and was not restricted to infected enterocytes. Ongoing treatment of infected piglets with a selective iNOS inhibitor resulted in significant increases in villous epithelial parasitism and oocyst excretion but was not detrimental to maintenance of mucosal barrier function. Intensified parasitism could not be attributed to attenuated fluid loss or changes in epithelial proliferation or replacement rate, inasmuch as iNOS inhibition did not alter severity of diarrhea, piglet hydration, Cl- secretion, or kinetics of bromodeoxyuridine-labeled enterocytes. These findings suggest that induction of iNOS represents a nonspecific response of the epithelium that mediates enterocyte defense against C. parvum infection. iNOS did not contribute to the pathogenic sequelae of C. parvum infection.

Nitric oxide synthase stimulates prostaglandin synthesis and barrier function in C. parvum-infected porcine ileum.

Cell culture models implicate increased nitric oxide (NO) synthesis as a cause of mucosal hyperpermeability in intestinal epithelial infection. NO may also mediate a multitude of subepithelial events, including activation of cyclooxygenases. We examined whether NO promotes barrier function via prostaglandin synthesis using Cryptosporidium parvum-infected ileal epithelium in residence with an intact submucosa. Expression of NO synthase (NOS) isoforms was examined by real-time RT-PCR of ileal mucosa from control and C. parvum-infected piglets. The isoforms mediating and mechanism of NO action on barrier function were assessed by measuring transepithelial resistance (TER) and eicosanoid synthesis by ileal mucosa mounted in Ussing chambers in the presence of selective and nonselective NOS inhibitors and after rescue with exogenous prostaglandins. C. parvum infection results in induction of mucosal inducible NOS (iNOS), increased synthesis of NO and PGE2, and increased mucosal permeability. Nonselective inhibition of NOS (NG-nitro-L-arginine methyl ester) inhibited prostaglandin synthesis, resulting in further increases in paracellular permeability. Baseline permeability was restored in the absence of NO by exogenous PGE2. Selective inhibition of iNOS [L-N6-(1-iminoethyl)-L-lysine] accounted for approximately 50% of NOS-dependent PGE2 synthesis and TER. Using an entire intestinal mucosa, we have demonstrated for the first time that NO serves as a proximal mediator of PGE2 synthesis and barrier function in C. parvum infection. Expression of iNOS by infected mucosa was without detriment to overall barrier function and may serve to promote clearance of infected enterocytes.