The Pig Olfactory Brain: A Primer.

Despite the fact that pigs are reputed to have excellent olfactory abilities, few studies have examined regions of the pig brain involved in the sense of smell. The present study provides an overview of the olfactory bulb, anterior olfactory nucleus, and piriform cortex of adult pigs using several approaches. Nissl, myelin, and Golgi stains were used to produce a general overview of the organization of the regions and confocal microscopy was employed to examine 1) projection neurons, 2) GABAergic local circuit neurons that express somatostatin, parvalbumin, vasoactive intestinal polypeptide, or calretinin, 3) neuromodulatory fibers (cholinergic and serotonergic), and 4) glia (astrocytes and microglia). The findings revealed that pig olfactory structures are quite large, highly organized and follow the general patterns observed in mammals.

Endoluminal vacuum therapy for gastrojejunal anastomotic leaks after Roux-en-Y gastric bypass: a pilot study in a swine model.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) consistently produces the most sustainable weight loss among common interventions for morbid obesity. Anastomotic leaks at the gastrojejunal (GJ) connection result in severe morbidity. We apply endoluminal negative pressure vacuum devices (EVD) to heal anastomotic leaks in a swine model. METHODS: RYGB was performed in 10 pigs (3 control, 7 experimental). GJ anastomoses were fashioned, and a 2-cm defect was made across the staple line. In controls, the defects remained open. In experimental pigs, the EVD was placed across the defect and kept at continuous 50 mmHg suction. All pigs were euthanized on postoperative day seven unless they displayed signs of peritonitis or sepsis. Fluoroscopy and necropsy were performed to assess a persistent leak, and tissue specimens were sent to histology to evaluate for degree of inflammation and ischemia. RESULTS: All three control pigs' GJ anastomoses demonstrated evidence of a persistent leak. All seven experimental pigs with the EVD in place showed evidence that their leak had sealed at time of fluoroscopy (p value 0.008). CONCLUSIONS: Endoluminal vacuum therapy is well tolerated in a swine model. GJ anastomotic leaks were consistently sealed with our device in place compared to controls. This therapy shows promise as a method to address GJ leaks in the bariatric population, and thus, we believe additional evaluation is warranted.

Extracellular adenosine regulates colitis through effects on lymphoid and nonlymphoid cells.

Adenosine is a purine metabolite that can mediate anti-inflammatory responses in the digestive tract through the A(2A) adenosine receptor (A(2A)AR). We examined the role of this receptor in the control of inflammation in the adoptive transfer model of colitis. Infection of A(2A)AR(-/-) mice with Helicobacter hepaticus increased colonic inflammation scores compared with uninfected A(2A)AR controls. Comparison of T cell subsets in wild-type and A(2A)AR(-/-) mice revealed differences in markers associated with activated helper T (Th) cells and regulatory T (Treg) cells. Previous studies showed that expression of A(2A)AR on CD45RB(HI) and CD45RB(LO) Th cells is essential for the proper regulation of colonic inflammation. Adoptive transfer of CD45RB(HI) with CD45RB(LO) from wild-type mice into RAG1(-/-)/A(2A)AR(-/-) mice induced severe disease within 3 wk, although transfer of the same subsets into RAG1(-/-) mice does not induce colitis. This suggests that the presence of A(2A)AR on recipient cells is also important for controlling colitis. To investigate the role of A(2A)AR in myeloid cells, chimeric recipients were generated by injection of bone marrow from RAG1(-/-) or RAG1(-/-)/A(2A)AR(-/-) mice into irradiated RAG1(-/-) mice. After adoptive transfer, these recipients did not develop colitis, regardless of A(2A)AR expression by the donor. Together, our results suggest that the control of inflammation in vivo is dependent on A(2A)AR signaling through multiple cell types that collaborate in the regulation of colitis by responding to extracellular adenosine.

Detection of pathogenic Batrachochytrium dendrobatidis using water filtration, animal and bait testing.

The pathogen Batrachochytrium dendrobatidis (Bd) can be challenging to detect at endangered amphibian reintroduction sites. Pre-release Bd detection can be confounded by imperfect animal sampling and the absence of animals. In Study 1, we used historical Bd-positive sites, to concurrently evaluate water filtrates and mouth bar (tadpoles) or skin swab (caudates) samples for Bd using molecular beacon realtime PCR. In Study 2, during a natural outbreak, we used PCR to detect Bd from zoospore-attracting keratin baits (three avian, three snake species). In Study 1, no captured animals (n=116) exhibited clinical signs, although 10.6% were positive, representing three of seven species sampled. In contrast, 5.4% of water filters (n=56) were Bd-positive. In Study 2, after short incubation times, a single duck down feather tested Bd-positive. In conclusion, Bd was detected in asymptomatic amphibians and water filtrate at two sites, and from water only, at two other sites. With continued refinement, semi-quantitative Bd water filtrate screening could better define zoospore-specific disease risk, allowing better characterization of the free-living phase of the organism's life cycle. Finally, these results suggest wild aquatic birds (e.g., waterfowl) should be systematically explored as a means of Bd spread. Since large numbers of aquatic birds migrate, even low Bd transfer rates could be a significant means for disease dissemination.

Spontaneous, immune-mediated gastric inflammation in SAMP1/YitFc mice, a model of Crohn's-like gastritis.

BACKGROUND & AIMS: Crohn's disease (CD) can develop in any region of the gastrointestinal tract, including the stomach. The etiology and pathogenesis of Crohn's gastritis are poorly understood, treatment approaches are limited, and there are not many suitable animal models for study. We characterized the features and mechanisms of chronic gastritis in SAMP1/YitFc (SAMP) mice, a spontaneous model of CD-like ileitis, along with possible therapeutic approaches. METHODS: Stomachs from specific pathogen-free and germ-free SAMP and AKR mice (controls) were evaluated histologically; the presence of Helicobacter spp was tested in fecal pellets by polymerase chain reaction analysis. In vivo gastric permeability was quantified by fractional excretion of sucrose, and epithelial tight junction protein expression was measured by quantitative reverse-transcription polymerase chain reaction analysis. The effects of a proton pump inhibitor (PPI) or corticosteroids were measured, and the ability of pathogenic immune cells to mediate gastritis was assessed in adoptive transfer experiments. RESULTS: SAMP mice developed Helicobacter-negative gastritis, characterized by aggregates of mononuclear cells, diffuse accumulation of neutrophils, and disruption of epithelial architecture; SAMP mice also had increased gastric permeability compared with controls, without alterations in expression of tight junction proteins. The gastritis and associated permeability defect observed in SAMP mice were independent of bacterial colonization and reduced by administration of corticosteroids but not a PPI. CD4(+) T cells isolated from draining mesenteric lymph nodes of SAMP mice were sufficient to induce gastritis in recipient SCID mice. CONCLUSIONS: In SAMP mice, gastritis develops spontaneously and has many features of CD-like ileitis. These mice are a useful model to study Helicobacter-negative, immune-mediated Crohn's gastritis.

Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity.

Long interspersed nuclear element-1 (L1)­mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNA­induced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNA­induced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.

Defining the Specific Pathogen-Free State of Xenopus Using TaqMan Assays.

Colonies of valuable inbred and transgenic laboratory-reared Xenopus frogs maintained for research constitute naïve populations of animals susceptible to some opportunistic infectious diseases. Therefore, it is prudent to characterize any new animal acquisitions before introduction into an existing colony as a biosecurity measure to preclude the concurrent introduction of an infectious microorganism associated with the new animal(s). In addition, some pathogens of Xenopus, such as Chlamydia and Mycobacterium spp, are zoonotic diseases, placing frog aquarists at risk for acquiring an infection. Because it is not cost effective to test for all diseases of Xenopus frogs, we have defined a subset of prevalent infectious microorganisms and developed TaqMan polymerase chain reaction (PCR) assays to detect these agents. The specific pathogens in our test panel were selected from relatively recent publications where they reportedly caused morbidity and/or mortality in Xenopus laevis and/or X. tropicalis The assays herein do not constitute a comprehensive list of infectious diseases of Xenopus frogs. Therefore, a frog devoid of the infectious agents in our test panel are characterized as "specific pathogen-free." Three of the described quantitative polymerase chain reaction (qPCR) assays detect many species within their genus (i.e., qPCRs for ranaviruses, Chlamydia spp, and Cryptosporidia spp).

Association of Mycoplasma corogypsi and Polyarthritis in a Black Vulture (Coragyps atratus) in Virginia.

On 10 October 2007, a Black Vulture (Coragyps atratus) was presented to the Wildlife Center of Virginia, Waynesboro, Virginia, USA, because of an inability to fly. Examination revealed multiple swollen, fluctuant joints. The bird suffered from lead toxicosis and had a prominent leukocytosis. Histopathologic evaluation revealed an acute fibrinoheterophilic polyarthritis, and results of routine aerobic and anaerobic culture of joint fluid were negative, although Mycoplasma sp. sequence-specific polymerase chain reaction was positive. Amplification of a portion of the 16S rRNA and subsequent phylogenetic analysis of the amplicon identified Mycoplasma corogypsi. This is the first report of polyarthritis being diagnosed in association with a Mycoplasma sp. in a vulture species. However, fulfilling Koch's postulates through experimental infections is required to draw conclusions concerning an etiologic diagnosis.

Critical role of bacterial dissemination in an infant rabbit model of bacillary dysentery.

The bacterial pathogen Shigella flexneri causes 270 million cases of bacillary dysentery (blood in stool) worldwide every year, resulting in more than 200,000 deaths. A major challenge in combating bacillary dysentery is the lack of a small-animal model that recapitulates the symptoms observed in infected individuals, including bloody diarrhea. Here, we show that similar to humans, infant rabbits infected with S. flexneri experience severe inflammation, massive ulceration of the colonic mucosa, and bloody diarrhea. T3SS-dependent invasion of epithelial cells is necessary and sufficient for mediating immune cell infiltration and vascular lesions. However, massive ulceration of the colonic mucosa, bloody diarrhea, and dramatic weight loss are strictly contingent on the ability of the bacteria to spread from cell to cell. The infant rabbit model features bacterial dissemination as a critical determinant of S. flexneri pathogenesis and provides a unique small-animal model for research and development of therapeutic interventions.

Molecular phylogeny of the pinworms of mice, rats and rabbits, and its use to develop molecular beacon assays for the detection of pinworms in mice.

Though pinworm infestation has been prevalent since the early years of laboratory animal medicine, the genomes of these parasites have not yet been sequenced. The authors used high-fidelity polymerase chain reaction to amplify a large portion of the ribosomal gene complex of four pinworm species commonly found in lab rodents and rabbits (Aspiculuris tetraptera, Passalurus ambiguus, Syphacia muris and Syphacia obvelata). They determined DNA sequences for these complexes and carried out phylogenetic analysis. Using this information, the authors developed real-time molecular beacon assays for pinworm detection, comparing the new diagnostic approach with traditional methods such as perianal tape testing, fecal flotation and direct examination of intestinal content.

A novel mycoplasma detected in association with upper respiratory disease syndrome in free-ranging eastern box turtles (Terrapene carolina carolina) in Virginia.

Clinical signs of upper respiratory tract disease-like syndrome (URTD-LS) were observed in free-ranging eastern box turtles (Terrapene carolina carolina) from Virginia, USA (May 2001-August 2003), some of which also had aural abscesses. After a Mycoplasma sp. was detected by polymerase chain reaction (PCR), a study was undertaken to better define the range of clinical signs of disease and to distinguish mycoplasma-associated URTD-LS from other suspected causes of URTD-LS and aural abscessation in box turtles. Nasal and/or ocular swabs (from turtles possessing URTD-LS) or nasal washes (from asymptomatic turtles) were collected from turtles May 2001-August 2003; samples were assayed for Mycoplasma spp., chelonian herpesvirus, and iridoviruses by PCR testing. A partial DNA sequence (933 bases) of the small ribosomal subunit (16S rRNA) of the box turtle Mycoplasma sp. was analyzed to determine its phylogenetic relatedness to other Mycoplasma spp. of veterinary interest. Mycoplasma sp. was detected in seven (six with clinical signs of URTD-LS; one asymptomatic) of 23 fortuitously collected animals from six of 11 Virginia counties. Clinical signs in Mycoplasma sp.-infected animals included unilateral to bilateral serous to mucopurulent nasal discharge, epiphora, ocular edema, and conjunctival injection. Five Mycoplasma sp.-positive animals possessed aural abscesses; two did not. Analysis of the mycoplasma 16S rRNA gene sequence from one asymptomatic and three symptomatic animals representing four counties revealed a consensus Mycoplasma sp. sequence closely related to, but distinct from, M. agassizii. None of the samples collected contained viral DNA of chelonian herpesviruses or invertebrate and vertebrate (including FV3) iridoviruses. In conclusion, a new Mycoplasma sp. was associated with URTD-LS in native box turtles from Virginia that was not codetected with other suspected causes of chelonian upper respiratory disease; there was no proof of a direct relationship between aural abscessation and the Mycoplasma sp.

Phylogenetic analysis of avian poxviruses among free-ranging birds of Virginia.

Polymerase chain reaction was used to amplify a portion of the avian poxvirus core 4b gene of infected free-ranging birds that presented at the Wildlife Center of Virginia during the 2003 and early 2004 years. The species of bird infected were a great blue heron (Ardea herodias), two American crows (Corvus brachyrhyncos), two American robins (Turdus migratorius), two mourning doves (Zenaida macroura), a red-tailed hawk (Buteo jamaicensis), a blue-gray gnatcatcher (Polioptila caerulea), a northern mockingbird (Mimus polyglottos), a house finch (Carpodacus mexicanus), and a northern cardinal (Cardinalis cardinalis). Phylogenetic analysis was performed using the consensus sequences determined for each avian case in Virginia in combination with avian poxvirus core 4b gene sequence from isolates previously described in Europe and that of vaccinia virus. Alignment of DNA sequences identified areas of point mutations and, in the case of a single mourning dove, the incorporation of a triplet of nucleotides. Maximum-likelihood analysis grouped the 2003-2004 Virginia avian poxviruses into a clade distinct from those reported in European free-ranging birds, with the exception of a single case in a mourning dove that clustered within one European clade. The cladogram that resulted from our analysis of the European isolates is in agreement with those previously published. This study identified a distinct clade of avian poxvirus unique from four clades previously described and associated with epornitics in free-ranging birds, where the core 4b gene DNA sequence has been the basis of comparison.

Successful rederivation of contaminated immunocompetent mice using neonatal transfer with iodine immersion.

There is an ongoing need to eradicate intercurrent disease from research mouse colonies. Commonly used surgical methods, however, are expensive and time-consuming. The purpose of this study was to determine the percentage of litters that could be rederived from infected mouse colonies by neonatal transfer. We immersed neonatal mice in a dilute iodine solution and transferred them to disease-free foster mothers within 48 h of birth. Donor and foster mothers were evaluated for pathogens by serology and fecal polymerase chain reaction (PCR) assay. Of 55 donor mothers, 100% were positive serologically and 59% were positive by fecal PCR for one or more tested organisms, including mouse hepatitis virus, Theiler's murine encephalomyelitis virus, mouse rotavirus, and Helicobacter hepaticus. At 4 to 6 weeks after neonatal transfer, 95% of foster mothers (which served as sentinels for the transferred pups) tested free of pathogens, the exceptions being one case of mouse parvovirus 1 and two of Helicobacter spp. We suggest that cross-fostering is a viable low-cost method for rederivation of mouse colonies contaminated with pathogens such as mouse hepatitis virus, Theiler's murine encephalomyelitis virus, mouse rotavirus, and H. hepaticus.

Phylogenetic classification of the frog pathogen Amphibiothecum (Dermosporidium) penneri based on small ribosomal subunit sequencing.

We determined 1,600 base pairs of DNA sequence in the 18S small ribosomal subunit from two geographically distinct isolates of Dermosporidium penneri. Maximum likelihood and parsimony analysis of these sequences place D. penneri in the order Dermocystida of the class Mesomycetozoea. The 18S rRNA sequences from these two isolates only differ within a single region of 16 contiguous nucleotides. Based on the distant phylogenetic relationship of these organisms to Amphibiocystidium ranae and similarity to Sphaerothecum destruens we propose the organism be renamed Amphibiothecum penneri.

The effects of cage design on airborne allergens and endotoxin in animal rooms: high-volume measurements with an ion-charging device.

Respiratory symptoms related to both endotoxins and animal allergens continue to be an important cause of occupational disease for animal technicians and scientists working with rodents. Better sampling methods for airborne allergens and endotoxin are needed to help standardize compliance with federal occupational health regulations. Using an ion-charging device, we sampled 20 mouse rooms and four rat rooms at the University of Virginia, along with 43 domestic living rooms in houses in the Charlottesville area with at least one cat or dog. The use of filter tops on cages corresponds to a 50-fold reduction in mean levels of both airborne allergens (P < 0.001) and endotoxin (P < 0.001). The use of vented cages with filtered exhaust ports was associated with additional reductions. However, the mean airborne endotoxin level in all rooms using filter tops without a filtered exhaust port on the cages was significantly lower (P = 0.003) than the level in domestic living rooms. Our results for maximum airborne allergens or endotoxin are comparable with previous reports. However, the sensitivity of the technique allows an accurate assessment of low-level exposure, which makes it possible to evaluate the effect of cage designs. In addition, this approach allows direct comparison with results for airborne allergen and endotoxin in domestic homes. The results could allow a more consistent approach to the application of occupational health guidelines.

Optical Imaging of Paramagnetic Bead-DNA Aggregation Inhibition Allows for Low Copy Number Detection of Infectious Pathogens.

DNA-paramagnetic silica bead aggregation in a rotating magnetic field facilitates the quantification of DNA with femtogram sensitivity, but yields no sequence-specific information. Here we provide an original description of aggregation inhibition for the detection of DNA and RNA in a sequence-specific manner following loop-mediated isothermal amplification (LAMP). The fragments generated via LAMP fail to induce chaotrope-mediated bead aggregation; however, due to their ability to passivate the bead surface, they effectively inhibit bead aggregation by longer 'trigger' DNA. We demonstrate the utility of aggregation inhibition as a method for the detection of bacterial and viral pathogens with sensitivity that approaches single copies of the target. We successfully use this methodology for the detection of notable food-borne pathogens Escherichia coli O157:H7 and Salmonella enterica, as well as Rift Valley fever virus, a weaponizable virus of national security concern. We also show the concentration dependence of aggregation inhibition, suggesting the potential for quantification of target nucleic acid in clinical and environmental samples. Lastly, we demonstrate the ability to rapidly detect infectious pathogens by utilizing a cell phone and custom-written application (App), making this novel detection modality fully portable for point-of-care use.

Components of gene therapy experimentation that contribute to relative risk.

Gene therapy is the purposeful delivery of genetic material to somatic cells for the purpose of treating disease or biomedical investigation. Either viral or non-viral vector methods can be used. The risk of collateral exposure of laboratory animal care personnel to gene therapy vectors is dependent on a number of factors. These factors are intrinsic to the gene therapy vector (the vehicle for genetic conveyance), product encoded by the genetic construct delivered, method of delivery, and immune status of the recipient. The component risks of gene therapy experiments can be analyzed to surmise the overall relative risk of the experiment. Knowledge of the components that contribute potential hazardous risk to a study can assist animal care staff in identifying area(s) where prudent practices should be focused. Gene therapy experiments involving viral vectors are generally performed at either biosafety level 2 or 3. The objective of this review is to report on various components of gene therapy experiments, focusing on characteristics of viral and non-viral vectors, to assist the laboratory animal science community in determining prudent biosafety practices.

Comparison of an espB gene fecal polymerase chain reaction assay with bacteriologic isolation for detection of Citrobacter rodentium infection in mice.

PURPOSE: To develop a polymerase chain reaction (PCR) assay for specific detection of Citrobacter rodentium in fecal samples of mice and to compare this assay with bacterial isolation and identification methods. METHODS: The target sequence of the PCR assay was the espB gene encoding a secreted virulence factor. To facilitate visual identification during primary isolation on MacConkey agar containing ampicillin, C. rodentium ATCC type strain 51459 was transformed by use of a plasmid encoding the enhanced green fluorescent protein (EGFP) and ampicillin resistance. The EGFP-C. rodentium was inoculated into Swiss Webster (SW) mice to study the time course of detection of the organism by use of fecal PCR analysis, bacterial isolation, and development of colonic hyperplasia by light microscopy. Lactose-fermenting fluorescent bacterial colonies identified during primary isolation of fecal bacteria on MacConkey-ampicillin agar were identified by use of biochemical typing. RESULTS: Mice inoculated with EGFP-transformed C. rodentium developed colonic mucosal hyperplasia, characterized by a three-fold increase in colonic crypt height that peaked at post-inoculation day (PID) 14. The espB PCR assay detected as little as 0.3 colony-forming units of C. rodentium. The PCR assay was specific in that it did not detect the espB gene of Escherichia coli 0157. Results of in vivo studies in SW mice indicated that EGFP-C. rodentium could be detected by use of espB fecal PCR analysis in 100% of inoculated mice tested on PID 1, 3, 7, and 8, in 60% on PID 9, and in 20% on PID 10 (n = 5). Bacterial isolation from the same fecal samples detected the organism in 100% of the inoculated mice on PID 7, in 50% on PID 8, and in none on subsequent PID 9-14. The ability of the PCR assay to detect C. rodentium in fresh feces of inoculated mice was significantly better than that of bacterial isolation methods (Fisher-Irwin exact test, P < 0.01). At the time of peak colonic hyperplasia, the organism could no longer be cultivated or detected in mice by use of fecal PCR analysis. CONCLUSIONS: The EGFP-C. rodentium was capable of inducing transmissible murine colonic hyperplasia similar to that previously reported in SW mice. The PCR assay for detection of the espB gene sequence of C. rodentium in total fecal DNA was a more sensitive diagnostic assay than was bacterial isolation.

Outbreak of otitis media caused by Burkholderia gladioli infection in immunocompromised mice.

An athymic nude mouse with severe head tilt due to otitis media was identified. Within weeks of identification of this first case, immune-deficient mice of various genotypes from the same facility were similarly affected, and cases from other facilities were found within two months. Culture of ear exudate specimens from affected mice yielded bacteria that were initially identified as Burkholderia cepacia, a plant pathogen considered an important opportunistic pathogen in persons with cystic fibrosis or chronic granulomatous disease. Several of these isolates, however, were subsequently identified as B. gladioli on the basis of results of biochemical analysis and a species-specific polymerase chain reaction (PCR) assay. Genotyping analysis revealed clonality among the isolates, indicating a shared strain among affected mice. A 16S rDNA-based PCR assay specific for the genera Burkholderia and Ralstonia, and a selective culture medium were used in efforts to characterize the epidemiology of this outbreak. In addition to culture of specimens from the oropharyngeal cavity of affected mice, samples were obtained from the environment, feces, sipper tubes, drinking water, and soiled bedding from cages of affected individuals. Burkholderia gladioli was most consistently detected in oropharyngeal swab specimens from affected mice. The PCR assay was equivalent to selective culture in identifying mice in the carrier state that did not have clinical signs of infection. However, neither detection method had sufficient sensitivity to reliably identify all carrier mice, causing the organism to persist at low levels unless entire colonies of immune-deficient mice were removed. The organism was highly resistant to antibiotic therapy. The source and epidemiology of this organism remain unknown. This epizootic serves as an important reminder that immunocompromised rodent colonies may harbor important human opportunistic pathogens.

Molecular phylogeny of Pseudocapillaroides xenopi (Moravec et Cosgrov 1982) and development of a quantitative PCR assay for its detection in aquarium sediment.

We used high-fidelity PCR to amplify a portion of the small ribosomal subunit (18S rRNA) of Pseudocapillaroides xenopi, a nematode that parasitizes the skin of Xenopus laevis. The 1113-bp amplicon was cloned, sequenced, and aligned with sequences from 22 other nematodes in the order Trichocephalida; Caenorhabditis elegans was used as the outgroup. Maximum-likelihood and Bayesian inference phylogenetic analyses clustered P. xenopi in a clade containing only members of the genus Capillaria. Our analyses support the following taxonomic relationships: 1) members of the family Trichuridae form a clade distinct from those in the family Trichocephalida; 2) members of the genera Trichuris and Capillaria form 2 distinct clades within the family Trichuridae; and 3) the genus Trichuris includes 2 distinct clades, one representing parasites that infect herbivores and the other representing parasites that infect omnivores and carnivores. Using 18S rRNA sequence unique to P. xenopi, we developed a Taq Man quantitative PCR assay to detect this P. xenopi sequence in total DNA isolated from aquarium sediment. The assay's lower limit of detection is 3 copies of target sequence in a reaction. The specificity of our assay was validated by using negative control DNA from 9 other pathogens of Xenopus. Our quantitative PCR assay detected P. xenopi DNA in the sediment of 2 of 12 aquaria from the source institution of the specimen used to develop the assay; these aquaria had been treated with ivermectin 6 mo previously.