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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.

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.

Typical enteropathogenic E. 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 mechanisms 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 purpose of this study was to (1) determine the genomic similarity between kitten aEPEC and human aEPEC isolates and (2) to 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 lethal infection (LI). Variation in adherence pattern 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 are 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.

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.

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.

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.

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.

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.

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 ≥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 small intestine is the principal site of nutrient and water absorption. These functions are served by a single layer of epithelial cells that are generated in intestinal crypts and migrate up mucosal villi whereupon they express digestive enzymes and nutrient transporters. The protozoal parasite, Cryptosporidium parvum, replicates within villus epithelial cells resulting in accelerated loss of enterocytes, villous atrophy, nutrient malabsorption, and severe diarrhea. Infection by Cryptosporidium is responsible for 6% of all diarrheal disease, 37.7% of recreational water-associated and 8.5% of drinking water-associated outbreaks of gastroenteritis of known and suspected infectious etiology (1, 2). The infection is responsible for 24% of cases of chronic diarrhea in people with HIV-AIDS worldwide (1, 3, 4) and up to 26% of cases of chronic diarrhea in children of developing countries (5). There are no consistently effective antimicrobial treatments or vaccines for Cryptosporidium infection. Recovery from infection depends upon innate and specific epithelial defense mechanisms that remain poorly understood and culminates in repopulation of the villi by functionally mature intestinal epithelium. These reparative mechanisms must outpace the life-threatening consequences of diarrheal dehydration and starvation. Accordingly, therapeutic approaches that promote epithelial defense and repair are likely to have a significant impact on the morbidity and mortality of C. parvum infection. The amino acid L-arginine has been demonstrated in several models of intestinal injury to promote epithelial defense and repair (6–10). These effects are attributed to metabolism of L-arginine to nitric oxide by nitric oxide synthase and conversion of L-arginine by arginase to L-ornithine, the precursor of polyamines. We and others have shown that in vivo, C. parvum infection results in epithelial induction of nitric oxide synthase II (iNOS) (11, 12). Knockout or pharmacological inhibition of iNOS activity results in significant increases in epithelial parasitism and oocyst excretion (11–13). These effects occur in the absence of alterations in epithelial turnover or secretion suggesting that nitric oxide exerts direct inhibitory effects on the parasite (11). Accordingly, exogenous nitric oxide has been shown in vitro to inhibit excystation of C. parvum sporozoites and reduce their viability (13). Nitric oxide is synthesized exclusively from L-arginine. In the neonate, milk is a poor source of L-arginine (14, 15) and the majority of L-arginine must be synthesized endogenously by the intestinal epithelium (16, 17). It is also presumed that severe villous atrophy, a key feature of C. parvum infection, culminates in an immature, crypt-like epithelium that is incapable of nutrient transport. We therefore surmised that L-arginine deficiency may contribute to poor epithelial defense of the newborn against C. parvum infection. Whether or not the intestinal epithelium is able to transport L-arginine in C. parvum infection has important clinical implications for use of oral L-arginine to promote epithelial defense. Four kinetically-defined systems are reportedly capable of L-arginine transport; y+, B0,+, bo,+, and y+L. Genes encoding the proteins responsible for the activity of system y+ and B0,+ have been cloned and designated as the cationic amino acid-specific transporters (CAT-1–4) (18–22) and ATB0,+ (23), respectively. In a variety of cell and tissue types, L-arginine transporters are coordinately induced with iNOS enabling extracellular L-arginine to be used preferentially for nitric oxide synthesis (24–27). We are not aware of any studies examining the ability of infected intestinal epithelium to transport luminal L-arginine, the specific transport system activities and transporter proteins responsible for L-arginine uptake by infected gut, or the influence of iNOS activity on epithelial transport of L-arginine. The C. parvum infected neonatal piglet is a unique experimental model that fully recapitulates human cryptosporidiosis, the pathophysiology of which is not reproduced in traditional animal models. In the present study we used intact mucosa from C. parvum infected neonatal piglets to demonstrate that infected ileum transports L-arginine at rates equivalent to that of uninfected epithelium despite the presence of profound villous atrophy. This could be attributed to enhanced uptake of L-arginine by individual epithelial cells in the infection. Unexpectedly, there were no differences in L-arginine transport system activities (y+ and B0,+) or level of transporter gene expression (CAT-1, CAT-2A, and ATB0,+) 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. Further, transport of L-arginine by the infected epithelium was significantly inhibited by pharmacological blockade of arginase activity. These results suggest that intracellular catabolism of L-arginine by arginase II facilitates the uptake of L-arginine through transport systems that do not differ from that of uninfected cells. Induction of arginase II has not been previously described for the intestinal epithelium and may limit nitric oxide synthesis by competing with iNOS for utilization of L-arginine or promote the use of L-arginine for the synthesis of reparative polyamines.

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 ≥ 10 μg/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.

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-κB 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.

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 ∼50% of NOS-dependent PGE2synthesis and TER. Using an entire intestinal mucosa, we have demonstrated for the first time that NO serves as a proximal mediator of PGE2synthesis 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.

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.

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 ≥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.

The small intestine is lined by a single layer of epithelial cells that are responsible for nutrient and water absorption and serve as the first line of defense against a hostile intraluminal environment. The protozoal parasite, Cryptosporidium parvum, replicates within and depletes intestinal epithelial cells resulting in progressive villous atrophy, nutrient malabsorption, and severe diarrhea. Infection by Cryptosporidium is responsible for 6% of all diarrheal disease, 37.7% of reported recreational water-associated and 8.5% of drinking water-associated outbreaks of gastroenteritis of known or suspected infectious etiology (1, 2). Cryptosporidiosis is responsible for diarrhea in 7 and 12% of children in developed and developing countries respectively (1, 3). There are no consistently effective antimicrobial treatments or vaccines for Cryptosporidium infection. Recovery depends upon the timely induction of epithelial defense mechanisms and replacement of deleted epithelial cells with functionally mature intestinal epithelium. These mechanisms must outpace the life-threatening consequences of diarrheal dehydration and starvation. Recent studies suggest that insufficient numbers of epithelial cells, rather than their immaturity, may be responsible for malabsorption, as C. parvum infected epithelial cells appear able to express amino acid and glucose transporters at levels comparable to uninfected epithelial cells (4)(author’s unpublished data). Accordingly, enteral therapies may represent feasible and practical avenues to promote epithelial defense and repair in C. parvum infection. Infectious enteritis of a variety of causes is associated with an increase in intestinal synthesis of nitric oxide (5). In C. parvum infected neonatal piglets and mice, intestinal epithelial cells are induced to express nitric oxide synthase II (iNOS) in vivo (6, 7). Knockout or pharmacological inhibition of iNOS activity results in significant increases in epithelial parasitism and oocyst excretion in C. parvum infection (6–8). Further, in vitro studies have shown a direct effect of nitric oxide on viability of C. parvum sporozoites (8). Nitric oxide is synthesized exclusively from the amino acid L-arginine. In adult mammals, L-arginine is synthesized by the proximal convoluted tubules of the kidney. In the newborn, renal synthesis of L-arginine is only minimally developed (9) and L-arginine is synthesized instead by the intestinal epithelial cells (9–11). Injury to the intestinal epithelium, increased amino acid requirements for growth and repair, and poor bio-availability of L-arginine in milk (10, 12, 13) may collectively predispose the neonate to L-arginine deficiency. In view of this, we have recently demonstrated significantly lower concentrations of L-arginine in intestinal mucosa of neonatal piglets with C. parvum infection compared to littermate controls (authors unpublished data). A significant role for L-arginine in neonatal gut defense is supported by studies where nitric oxide synthesis inhibitors extended the age to which neonatal mice remain susceptible to C. parvum infection (7) and identification of L-arginine deficiency as a risk factor for development of necrotizing enterocolitis (4, 14–21). Accordingly, oral delivery of L-arginine may be useful to promote nitric oxide synthesis and epithelial defense in C. parvum infected neonates. In addition to the anti-parasitic effects of nitric oxide, we have also demonstrated that nitric oxide stimulates the synthesis of endogenous prostaglandins by C. parvum infected neonatal intestine (22). Endogenous prostaglandins promote the maintenance of intestinal barrier function while at the same time inhibit neutral NaCl absorption and stimulate epithelial chloride secretion (23, 24). Consequently, promoting the synthesis of nitric oxide in C. parvum infection may worsen the severity of secretory diarrhea. The purpose of the present study was to determine if oral supplementation with L-arginine could augment nitric oxide synthesis and promote epithelial defense in neonatal piglets infected with C. parvum. The neonatal piglet is a highly valuable and unique experimental model that fully recapitulates human cryptosporidial infection, the pathophysiology of which is not reproduced in traditional laboratory animals (25).

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.

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.

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.

Critical to the success of HIV-1 subunit vaccines is the development of strategies to augment vaccine immunogenicity. Successful adjuvants must not only improve immunogenicity above current adjuvant levels, but must also decrease the dose of immunogen required for optimal immunogenicity. We have evaluated activated alpha2-macroglobulin (alpha2M*) and a squalene-based stable emulsion containing monophosphoryl lipid A (MPL-SE) with granulocyte-macrophage colony stimulating factor (GM-CSF) as adjuvants to enhance the immunogencity of candidate HIV immunogens. Balb/c mice were subcutaneously immunized on days 0, 14 and 28 with 100-0.1 microg of HIV-1 envelope gp120 C4-V3 immunogens from either HIV IIIB (C4-V3(IIIB)) or SHIV 89.6P (C4-V3(89.6P)). Immunogens were tested covalently coupled to alpha2M*, formulated with MPL-SE/GM-CSF, or as a combination of both. Using CFA/IFA, only 50 and 100 microg, but not lower doses of C4-V3(IIIB) peptides, induced antibody responses. In contrast, peak antibody responses were detected in mice immunized with 10 microg of C4-V3 peptide coupled to alpha2M* (alpha2M*-peptide). Similar to CFA/IFA, MPL-SE/GM-CSF induced optimal antibody responses at 50 and 100 microg of C4-V3 immunogen. However, the combination of MPL-SE/GM-CSF with alpha2M*-C4-V3 peptide decreased the dose of C4-V3 required for optimal response to 5 microg for C4-V3(IIIB), and to 0.1 microg for C4-V3(89.6P). Taken together, HIV envelope gp120 C4-V3 peptides covalently complexed with alpha2M* and formulated with MPL-SE/GM-CSF resulted in a subunit HIV immunogen capable of inducing anti-HIV envelope antibody responses at doses up to 100-fold less than those needed with CFA/IFA or MPL-SE/GM-CSF alone.

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.

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 mg/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.