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The murine bacterial pathogen Chlamydia muridarum (Cm) has been used to study human Chlamydia infections in various mouse models. CD4+ T-cells, natural killer cells, and interferon-gamma (IFN-γ)–mediated immunity are important to control experimentally induced Cm infections. Despite its experimental use, natural infection by Cm has not been documented in laboratory mice since the 1940s. In 2022, the authors reported the discovery of natural Cm infections in numerous academic institutional laboratory mouse colonies around the globe. To evaluate the impact of Cm infection in severely immunocompromised mice, 19 NOD.Cg- Prkdc scid Il2rg tm1Wjl/SzJ (NSG) mice were cohoused with Cm shedding, naturally infected immunocompetent mice and/or their soiled bedding for 4 weeks and subsequently euthanized. Clinical disease, characterized by lethargy, dyspnea, and weight loss, was observed in 11/19 NSG mice, and 16/18 NSG mice had neutrophilia. All mice exhibited multifocal to coalescing histiocytic and neutrophilic bronchointerstitial pneumonia (17/19) or bronchiolitis (2/19) with intraepithelial chlamydial inclusions (CIs). Immunofluorescence showed CIs were often associated with bronchiolar epithelium. CIs were frequently detected by immunohistochemistry in tracheal and bronchiolar epithelium (19/19), as well as throughout the small and large intestinal epithelium without lesions (19/19). In a subset of cases, Cm colonized the surface epithelium in the nasopharynx (16/19), nasal cavity (7/19), and middle ear canal (5/19). Endometritis and salpingitis with intraepithelial CI were identified in a single mouse. These findings demonstrate that Cm infection acquired through direct contact or soiled bedding causes significant pulmonary pathology and widespread intestinal colonization in NSG mice. [ABSTRACT FROM AUTHOR]

An otherwise healthy two-month-old female C57BL/6J mouse presented with a left-sided head tilt. Differential diagnoses included idiopathic necrotizing arteritis, bacterial otitis media/interna (Pasteurella pneumotropica, Pseudomonas aeruginosa, Streptococcus sp., Mycoplasma pulmonis and Burkholderia gladioli), encephalitis, an abscess, neoplasia, a congenital malformation and an accidental or iatrogenic head trauma. Magnetic resonance imaging (MRI) revealed a large space-occupying right olfactory lobe intra-axial lesion with severe secondary left-sided subfalcine herniation. Following imaging, the animal was euthanized due to poor prognosis. Histopathologic examination revealed a unilateral, full-thickness bone defect at the base of the cribriform plate and nasal conchae dysplasia, resulting in the herniation of the olfactory bulb into the nasal cavity. There was also a left midline-shift of the frontal cortex and moderate catarrhal sinusitis in the left mandibular sinus. The MRI and histopathologic changes are consistent with a congenital malformation of the nasal cavity and frontal aspect of the skull known as an ethmoidal meningoencephalocele. Encephaloceles are rare abnormalities caused by herniation of contents of the brain through a defect in the skull which occur due to disruption of the neural tube closure at the level anterior neuropore or secondary to trauma, surgical complications, cleft palate or increased intracranial pressure. The etiology is incompletely understood but hypotheses include genetics, vitamin deficiency, teratogens, infectious agents and environmental factors. Ethmoidal encephaloceles have been reported in multiple species including humans but have not been reported previously in mice. There are multiple models for spontaneous and induced craniofacial malformation in mice, but none described for ethmoidal encephaloceles. [ABSTRACT FROM AUTHOR]

Chlamydia muridarum (Cm) has reemerged as a moderately prevalent infectious agent in research mouse colonies. Despite its' experimental use, few studies evaluate Cm's effects on immunocompetent mice following its natural route of infection. A Cm field isolate was administered (orogastric gavage) to 8-week-old female BALB/cJ (C) mice. After confirming shedding (through 95d), these mice were cohoused with naive C57BL/6J (B6), C, and Swiss (J:ARC[S]) mice (n=28/strain) for 30 days. Cohoused mice (n=3-6 exposed and 1-6 control/strain) were evaluated 7, 14, 21, 63, 120, and 180 days post-cohousing (DPC) via hemograms, serum biochemistry analysis, fecal qPCR, histopathology, and Cm MOMP immunohistochemistry. Immunophenotyping was performed on spleen (B6, C, S; n=6/strain) and intestines (B6; n=6) at 14 and 63 DPC. Serum cytokine concentrations were measured (B6; n=6 exposed and 2 control) at 14 and 63 DPC. All B6 mice were shedding Cm by 3 through 180 DPI. One of 3 C and 1 of 6 S mice began shedding Cm at 3 and 14 DPC, respectively, with the remaining shedding thereafter. Clinical pathology was nonremarkable. Minimal-to-moderate enterotyphlocolitis and gastrointestinal associated lymphoid tissue (GALT) hyperplasia was observed in 15 and 47 of 76 Cm-infected mice, respectively. Cm antigen was frequently detected in GALT-associated surface intestinal epithelial cells. Splenic immunophenotyping revealed increased monocytes and shifts in T cell population subsets in all strains/timepoints. Gastrointestinal immunophenotyping (B6) revealed sustained increases in total inflammatory cells and elevated cytokine production in innate lymphoid cells and effector T cells (large intestine). Elevated concentrations of pro-inflammatory cytokines were detected in the serum (B6). Results demonstrate that while clinical disease was not appreciated, 3 commonly utilized strains of mice are susceptible to chronic enteric Cm infection which may alter various immune responses. Considering the widespread use of mice to model GI disease, institutions should consider excluding Cm from their colonies.

Chlamydia muridarum (Cm) has reemerged as a prevalent bacterial contaminant of academic research mouse colonies. A study was conducted to assess the effectiveness of husbandry and cage sanitization methods in preventing intercage transmission of Cm. To assess intercage transmission during cage change, a cage housing 2 Cm-free Swiss Webster (SW; Tac:SW) sentinel mice was placed randomly on each of 12 individually ventilated cage racks, housing cages with Cm-shedding mice, located in one of 2 animal holding rooms. Husbandry staff blinded to the study cages changed all cages in the animal holding rooms weekly using a microisolation cage technique. PCR testing performed at 180 d postplacement confirmed all mice remained negative for Cm. To assess the effectiveness of cage sanitization to eliminate Cm, we investigated transmission of Cm to a naive Cm-free SW and NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mouse cohoused for 7 d (repeated weekly for 4 wk) in cages assigned to one of 3 groups ( n = 10 pairs of mice/group). Cages that previously housed 2 Cm-shedding BALB/c mice were either washed in a tunnel washer (82.2 °C [180 °F] final rinse for an average of 16 s per run; n = 10) with and without postwashing autoclaving (121 °C for 20 min; n = 10), or were untreated (bedding change only; n = 10). Pre- and postsanitization swabs of each cage were assayed for Cm by PCR. All pretreatment swabs tested positive, while posttreatment swabs from all cages (excluding bedding change) tested negative. All SW and NSG mice, irrespective of group, remained negative for Cm as determined by PCR. These findings suggest that infectious Cm does not persist in untreated cages or after mechanical washing with and without autoclaving. Collectively, these findings suggest that neither our husbandry protocols nor inadequate cage sanitization methods likely contributed to the observed prevalence of Cm in contemporary research mouse colonies.

Chlamydia muridarum (Cm) is a moderately prevalent, gram-negative, intracellular bacterium that affects laboratory mice, causing subclinical to severe disease, depending on the host's immune status. The effectiveness of various antibiotic regimens aimed at eradicating Cm in both immunodeficient and immunocompetent laboratory mice was evaluated. NSG mice were cohoused with Cm-shedding BALB/cJ mice for 14 days to simulate natural exposure. Four groups of 8 infected NSG mice were treated for 7 days with either 0.08% sulfamethoxazole and 0.016% trimethoprim (TMS) in water, 0.0625% doxycycline in feed, 0.124%/0.025% TMS in feed, or 0.12% amoxicillin in feed. A control group was provided standard water and feed. The impact of treatment on gastrointestinal microbiota (GM) was performed using next-generation shotgun sequencing on the last day of treatment. TMS and Amoxicillin had negligible effects on GM, while doxycycline had the largest effect. All antibiotic treated NSG mice exhibited clinical disease, including dehydration, hunched posture, >20% weight loss, and dyspnea, leading to euthanasia 21-40 days post-treatment (32.6 ± 4.2 days; mean ± SD). Untreated controls were euthanized 14-33 days post-exposure (23.75 ± 5.9 days). All mice were fecal PCR positive for Cm at euthanasia. Histological evaluation revealed multifocal histiocytic and neutrophilic bronchointerstitial pneumonia and/or bronchiolitis featuring prominent intralesional chlamydial inclusion bodies in all mice. Subsequently, groups of 8 C57BL/6J, BALB/cJ, NOD.SCID, and NSG mice infected with Cm were treated with 0.124%/0.025% TMS in feed for 7 (BALB/cJ and C57BL/6J) or 21 days (NSG and NOD.SCID). All immunocompetent and NOD.SCID mice were negative for Cm by PCR 14 days post-treatment, remained clinically normal and had no evidence of Cm infection at necropsy, all NSG mice remained Cm positive and were euthanized. While these findings highlight the difficulties in eradicating Cm from highly immunodeficient mice, eradication of Cm from immunocompetent or moderately immunocompromised mice with antibiotics is feasible.

Mice are commonly infected with Nippostrongylus brasiliensis (Nb) to study their immune responses. However, biosecurity measures have not been established for housing Nb-infected mice and rats. Transmission reportedly does not occur when infected mice are cohoused with naive mice. To test this, we inoculated female NOD. Cg- Prkdc scid Il2rg tm1Wjl /Sz(NSG; n = 12) and C57BL/6J (B6; n = 12) mice with 750 Nb L₃ larvae. These mice were then cohoused with naïve NSG ( n = 24) and B6 ( n = 24) mice (1 infected and 2 naïve mice per cage (24 cages) for 28 d in static microisolation cages that were changed every 14 d. We also did several studies to determine the conditions that favor horizontal transmission. First, we assessed in vitro development to the L₃ stage of Nb egg-containing fecal pellets maintained under 4 environmental conditions (dry, moist, soiled bedding, and control). Second, we assessed infection of naïve NSG mice ( n = 9) housed in microisolation cages that contained soiled bedding spiked with infective L₃ larvae (10,000/cage). Third, we gavaged NSG mice ( n = 3) with Nb eggs to model the potential for infection after coprophagy. We found that naïve NSG (9 of 24) and B6 (10 of 24) mice cohoused with an infected cagemate passed Nb eggs in feces as early as 1 d after cohousing and intermittently thereafter for varying periods. This shedding was presumably the result of coprophagy because adult worms were not detected in the shedding mice at euthanasia. Although eggs developed in vitro into L₃ larvae under moist and control environmental conditions, none of the NSG mice housed in cages with L₃ -spiked bedding or gavaged with eggs became infected with Nb. These findings indicate that infectious horizontal transmission does not occur when mice are housed with Nb-shedding cage mates in static microisolation cages with a 14-d cage-changing interval. Results from this study can be used to inform biosecurity practices when working with Nb-infected mice.

Mouse kidney parvovirus (MKPV) causes inclusion body nephropathy in severely immunocompromised mice and renal interstitial inflammation in immunocompetent mice. Here we sought to determine the effects of MKPV on pre-clinical murine models that depend on renal function. To assess the effects of MKPV infection on the pharmacokinetics of 2 renally excreted chemotherapeutic agents, methotrexate and lenalidomide, we measured drug concentrations in the blood and urine of MKPV-infected or uninfected immunodeficient NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ (NSG) and immunocompetent C57BL/6NCrl (B6) female mice. No differences in plasma pharmacokinetics were observed for lenalidomide. However, the AUC of methotrexate was 1.5-fold higher in uninfected NSG mice compared with infected NSG mice, 1.9-fold higher in infected B6 mice compared with uninfected B6 mice, and 4.3-fold higher in uninfected NSG mice compared with uninfected B6 mice. MKPV infection did not significantly affect the renal clearance of either drug. To assess effects of MKPV infection on the adenine diet model of chronic kidney disease, MKPV-infected and uninfected B6 female mice were fed a 0.2% adenine diet, and clinical and histopathologic features of disease were assessed over 8 wk. MKPV infection did not significantly alter urine chemistry results, hemogram findings, or serum concentrations of BUN, creatinine, or symmetric dimethylarginine. However, infection did influence histologic outcomes. As compared with uninfected mice, MKPV-infected mice had more interstitial lymphoplasmacytic infiltrates after 4 and 8 wk of diet consumption and less interstitial fibrosis at week 8. Macrophage infiltrates and renal tubular injury were similar between in infected and uninfected mice. These findings indicate that MKPV infection had minimal effects on the renal excretion of 2 chemotherapeutics and on serum biomarkers of renal function. However, infection significantly influenced two histologic features of the adenine diet model of chronic renal disease. MKPV-free mice are critically important in studies evaluating renal histology as an experimental outcome.

Mouse kidney parvovirus (MKPV), a newly identified parvovirus of the genus Chaphamaparvovirus , causes inclusion body nephropathy in severely immunocompromised mice and is prevalent in research mouse colonies. As nonenveloped viruses, mammalian parvoviruses are stable and generally resist thermal inactivation; however, as a novel and highly divergent parvovirus, the thermal stability of MKPV is undefined. This study aimed to evaluate the ability of cage sanitization in a mechanical washer to eliminate MKPV. Cages contaminated by MKPV-infected mice were assigned to 1 of 3 treatment groups: 1) control (bedding change only); 2) sanitization in a tunnel washer (88°C final rinse for 20 s); or 3) sanitization in a tunnel washer followed by autoclave sterilization (121 °C for 20 min). The presence of MKPV on the cage's interior surface was assessed by PCR of cage swab extracts collected before and after cage treatment. After treatment and swabbing, each cage housed 4 MKPV-negative CD1 mice. Each group of naive CD1 mice was assigned to one of the treatment groups and was housed in a cage from this group for two, 1 wk periods. At 12, 17, and 20 wk after the first exposure, renal tissue was collected from 1 test mouse per cage and assessed for MKPV by PCR. MKPV was detected by PCR on the surface of 63% of the pretreatment cages. All cages sanitized in a tunnel washer with or without sterilization were PCR negative after treatment. Seven of 10 mice housed in untreated cages contained a mouse positive for MKPV by 20 wk after exposure. None of the mice housed in cages sanitized in a tunnel washer with or without sterilization tested positive for MKPV at any time point. This study indicates that MKPV contaminated caging can result in MKPV infection of mice, and the use of a tunnel washer at the temperature and duration evaluated was sufficient to remove MKPV nucleic acid and prevent MKPV transmission.

Demodex musculi is a prostigmatid follicular mite that has rarely been reported in laboratory mice. Although prevalence of this species has not been assessed formally, we have found that many imported mouse strains from noncommercial sources harbor Demodex mites. To assess whether an acaricide can be used to eradicate this mite, infested immunocompromised mice were provided ivermectin-compounded (12 ppm) feed without restriction for 8 wk (n = 10), were treated topically with moxidectin and imidacloprid (MI; 3 and 13 mg/kg, respectively) weekly for 8 wk (n = 10), or remained untreated (n = 10). Mice were confirmed to be mite-infested before treatment and were tested after treatment by using fur plucks (FP), deep skin scrapes (DSS), and PCR analysis of fur swabs. In addition, the presence of mites was confirmed through skin biopsies at 2 study endpoints (1 wk [n = 5] and 12 wk [ n = 5] after treatment). Samples collected before treatment and from untreated mice were positive for D. musculi at all time points and by all test modalities. After treatment, all ivermectin-treated animals remained infested, whereas mice treated with MI were repeatedly negative by all test modalities. An additional shortened treatment trial revealed that 4 wk of MI (n = 7) was insufficient to eradicate mites. Neither treatment produced any evidence of adverse effects according to hematology, serum chemistry parameters, behavior, body weight, and histopathology. Of the 70 PCR assays performed in treated mice, 14 were positive when FP+DSS was negative. In 6 cases where PCR results were negative, 5 were positive by FP+DSS and a single sample was positive on skin biopsy. Although PCR analysis has a high detection rate for D. musculi, FP+DSS can further enhance the detection rate. In conclusion, topical MI administered for 8 consecutive weeks can safely eradicate D. musculi from an immunocompromised mouse strain.

Demodex mites are microscopic, cigar-shaped, follicular mites often regarded as commensal microfauna in mammals. Although Demodex spp. can cause dermatologic disease in any immunocompromised mammal, they are rarely reported in laboratory mice. Recent identification of Demodex musculi in a colony of immunodeficient mice with dermatitis afforded us the opportunity to investigate the comparative sensitivity of 4 antemortem diagnostic techniques to detect D. musculi-superficial skin scrape (SSS), tape impression (TI), fur pluck (FP), and deep skin scrape (DSS)-which we performed on 4 anatomic sites (face, interscapular region [IS], caudal ventrum [CV], and caudal dorsum [CD]) in 46 mice. DSS had an overall detection rate of 91.1% (n = 112 tests), with the highest detection rates in IS (93.5%), CV (89.1%), and CD (90.0%). The detection rates for SSS (62.5%; n = 112 tests), TI (57.5%; n = 138 tests), and FP (62.7%; n = 158 tests) were all lower than for DSS. IS was the most reliable site. Results from combined FP and DSS samples collected from IS and CV yielded 100% detection, whereas the face was not a desirable sampling site due to inadequate sample quality and low detection rate. Demodex eggs and larvae were observed from FP more often than DSS (19.0% of 158 tests compared with 14.3% of 112 tests). In a subset of samples, an 18S rRNA PCR assay was equivalent to DSS for detection of mites (both 100%, n = 8). We recommend collecting samples from both IS and CV by both FP and DSS to assess for the presence of D. musculi and performing further studies to assess whether PCR analysis can be used as a diagnostic tool for the detection of Demodex mites in laboratory mice.

We evaluated PCR testing of filter tops from cages maintained on an IVC system through which exhaust air is filtered at the cage level as a method for detecting parasite-infected and -infested cages. Cages containing 4 naïve Swiss Webster mice received 360 mL of uncontaminated aspen chip or α-cellulose bedding (n = 18 cages each) and 60 mL of the same type of bedding weekly from each of the following 4 groups of cages housing mice infected or infested with Syphacia obvelata (SO), Aspiculuris tetraptera (AT), Myocoptes musculinus (MC), or Myobia musculi (MB) and Radfordia affinis (RA; 240 mL bedding total). Detection rates were compared at 30, 60, and 90 d after initiating bedding exposure, by using PCR analysis of filter tops (media extract and swabs) and testing of mouse samples (fur swab [direct] PCR testing, fecal flotation, anal tape test, direct examination of intestinal contents, and skin scrape). PCR testing of filter media extract detected 100% of all parasites at 30 d (both bedding types) except for AT (α-cellulose bedding, 67% detection rate); identified more cages with fur mites (MB and MC) than direct PCR when cellulose bedding was used; and was better at detecting parasites than all nonmolecular methods evaluated. PCR analysis of filter media extract was superior to swab and direct PCR for all parasites cumulatively for each bedding type. Direct PCR more effectively detected MC and all parasites combined for aspen chip compared with cellulose bedding. PCR analysis of filter media extract for IVC systems in which exhaust air is filtered at the cage level was shown to be a highly effective environmental testing method.

There is no consensus regarding the best practice for detecting murine pinworm infections. Initially, we evaluated 7 fecal concentration methods by using feces containing Aspiculuris tetraptera (AT) eggs (n = 20 samples per method). Sodium nitrate flotation, sodium nitrate centrifugation, Sheather sugar centrifugation, and zinc sulfate centrifugation detected eggs in 100% of samples; zinc sulfate flotation and water sedimentation detected eggs in 90%. All had better detection rates than Sheather sugar flotation (50%). To determine optimal detection methods, Swiss Webster mice were exposed to Syphacia obvelata (SO; n = 60) or AT (n = 60). We compared the following methods at days 0, 30, and 90, beginning 21 or 28 d after SO and AT exposure, respectively: fecal concentration (AT only), anal tape test (SO only), direct examination of intestinal contents (cecum and colon), Swiss roll histology (cecum and colon), and PCR analysis (pooled fur swab and feces). Detection rates for SO-exposed mice were: PCR analysis, 45%; Swiss roll histology, 30%; intestinal content exam, 27%; and tape test, 27%. The SO detection rate for PCR analysis was significantly greater than that for the tape test. Detection rates for AT-exposed mice were: intestinal content exam, 53%; PCR analysis, 33%; fecal flotation, 22%; and Swiss roll histology, 17%. The AT detection rate of PCR analysis combined with intestinal content examination was greater than for PCR analysis only and the AT detection rate of intestinal content examination was greater than for Swiss roll histology. Combining PCR analysis with intestinal content examination detected 100% of infected animals. No single test detected all positive animals. We recommend combining PCR analysis with intestinal content examination for optimal pinworm detection.

Fur mite outbreaks remain a persistent problem in laboratory mouse colonies. All currently published treatment methods are labor-intensive, expensive, or unreliable. During a recent outbreak with Myobia musculi and Myocoptes musculinus in a large colony (approximately 30,000 cages), we developed a feed-based treatment regime in which ivermectin was the active ingredient. Rodent feed was compounded with 3 different concentrations of ivermectin (12, 24, and 48 ppm) and γ-irradiated. Postcompounding analysis revealed loss of ivermectin during manufacturing, but the remaining drug was stable for at least 6 mo. In an 8-wk toxicity study in a C57BL/6NTac mouse breeding colony, ad-libitum feeding of the 3 diets yielded estimated doses of 1.3, 2.7, and 5.4 mg/kg. Adult mice lacked adverse clinical effects, except that 1 of the 144 mice in the 48-ppm group developed tremors and ataxia and was euthanized. No significant differences between doses were revealed by CBC, serum chemistry, body weight, or gross necropsy. Plasma drug concentrations plateaued at a dose-dependent level 7 to 10 d after initiation of treatment and decreased to undetectable levels 6 to 9 d after its discontinuation. Fertility of the P0 generation was unaffected. Pup mortality was higher in the 24- and 48-ppm groups, reaching 100% at the higher dose. Animals exposed to ivermectin as neonates had normal weaning weights, but mice receiving 24-ppm feed had lower adult weights. Our results indicate that using feed containing 12 ppm ivermectin (estimated ingested dose, 1.3 mg/kg) was safe in a C57BL/6NTac breeding colony.

We determined the efficacy of ivermectin-compounded feed against fur mites in mice and describe its use to eradicate mites in vivaria holding approximately 30,000 cages. C57BL/6NCrl mice infested with Myobia musculi and Myocoptes musculinus were treated with ivermectin-compounded feed (approximate ingested dose, 1.3 mg/kg) for 1, 4, or 8 consecutive weeks. Regardless of treatment duration, all treated mice, as well as contact sentinels, remained free of fur mites for as long as 21 wk after treatment. No adverse effects were observed. Subsequently, facility-wide treatment was implemented in an attempt to eradicate fur mites from 3 vivaria housing approximately 120,000 mice. Medicated feed was provided for 8 wk to ensure that all cages and mice were treated. A single investigative group reported adverse effects in their colony 4 wk after treatment was initiated; mortality was attributed to ivermectin toxicity after an intracranial injection at 1 d of age. Naïve pups were unaffected. No other adverse effects were noted. Approximately 14,500 skin scrape samples were evaluated during the 12-mo posttreatment surveillance period. All samples were negative for mites. To our knowledge, this is the first report of successful eradication of fur mites from a mouse colony of this large size.

Fur mites are a persistent problem in contemporary laboratory mouse colonies. We conducted several studies to evaluate fur mite diagnostic methodologies and interpretation of results. Retrospective analysis of test results from sentinel mice exposed to soiled bedding collected from colonies infested with Myobia musculi and Myocoptes musculinus revealed the skin scrape test to be more reliable than pelt examination, provided that both the head and dorsal thoracolumbar regions were sampled. To assess their diagnostic accuracy, 3 commercial laboratories were sent positive control slides containing mites, mite parts, or eggs in sets of slides containing diagnostic skin scrapings in varying ratios. Laboratory B correctly identified the positive control slide. Laboratory A identified 1 of 3 positive control slides, whereas laboratory C failed to identify both positive control slides submitted. To determine the time required for a mouse to shed its entire hair coat, fur of Crl:CD1(ICR), BALB/cAnNCrl, and Crl:CFW(SW) albino mice was dyed black and the presence of dyed fur evaluated monthly for 8 mo. Limited dyed hair was still present at 8 mo; therefore, finding eggs or egg casings many months after treatment cessation does not necessarily imply treatment failure. To evaluate the effectiveness of soiled bedding sentinels for detection of fur mites in a mite-infested colony, we exposed naïve mice to varying amounts (100%, 50%, 25%, 2.5%, and 0%) of soiled bedding in clean bedding. As little as 2.5% soiled bedding resulted in detection of a positive sentinel within a 2-mo period.