The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease Assay

Real-time polymerase chain reaction (PCR) is the current method of choice for detection and quantification of nucleic acids, especially for molecular diagnostics. Complementarity between primers and template is often crucial for PCR applications, as mismatches can severely reduce priming efficiency. However, little quantitative data on the effect of these mismatches is available. We quantitatively investigated the effects of primer-template mismatches within the 3′-end primer region on real-time PCR using the 5′-nuclease assay. Our results show that single mismatches instigate a broad variety of effects, ranging from minor (<1.5 cycle threshold, eg, A–C, C–A, T–G, G–T) to severe impact (>7.0 cycle threshold, eg, A–A, G–A, A–G, C–C) on PCR amplification. A clear relationship between specific mismatch types, position, and impact was found, which remained consistent for DNA versus RNA amplifications and Taq/Moloney murine leukemia virus versus rTth based amplifications. The overall size of the impact among the various master mixes used differed substantially (up to sevenfold), and for certain master mixes a reverse or forward primer-specific impact was observed, emphasizing the importance of the experimental conditions used. Taken together these data suggest that mismatch impact follows a consistent pattern and enabled us to formulate several guidelines for predicting primer-template mismatch behavior when using specific 5-nuclease assay master mixes. Our study provides novel insight into mismatch behavior and should allow for more optimized development of real-time PCR assays involving primer-template mismatches.During the past decade, real-time polymerase chain reaction (PCR) has established itself as an essential technique for reliable detection and quantification of nucleic acids.^1,2,3 The result is a widespread application of real-time PCR assays in both research^1,2,3,4 and diagnostic^4,5,6 laboratories. Vital to the specificity, sensitivity, and efficiency of real-time PCR are the primers. The most important primer characteristics contributing to a successful amplification are primer-template association and dissociation kinetics, possible secondary structures, and primer-template complementarity (Watson-Crick base-pairing).^7,8 Full complementarity between primer and template sequences is generally considered crucial for the specific amplification of a nucleic acid sequence, but can be difficult to achieve, in particular for applications depending on highly heterogenic nucleic acid input for amplification (eg, diagnostic assays for influenza virus and human immunodeficiency virus). Conserved regions are often too small to accommodate a typical real-time PCR assay (50 to 150 bp), exhibit inferior G-C contents or are prone to the formation of secondary structures. Primer-template mismatches can therefore be unavoidable.Unfortunately, mismatches between primers and template are known to affect both the stability of the primer-template duplex and the efficiency with which the polymerase extends the primer,^{7,8,9,10,11,12,13} potentially leading to biased results or even PCR failure.^14,15 Even apparently small effects on nucleic acid quantification (0.5 to 1.0 log underestimation of initial copy number) can have serious consequences, as illustrated by studies on the relation between viral load and disease prognosis in HIV-1.¹⁶The detrimental effects of primer-template mismatches can however also prove beneficial. They provide a discriminative force that can be used for PCR assays opting to distinguish between different nucleic acids (eg, single nucleotide polymorphism detection, allele-specific PCR), which have become important tools for modern molecular diagnostics.⁴Every mismatch, irrespective of its location within the primer sequence, will result in a decreased thermal stability of the primer-template duplex, thus potentially affecting PCR specificity. However, mismatches located in the 3′ end region (defined as the last 5 nucleotides of the 3′ end region) of a primer have significantly larger effects on priming efficiency than more 5′ located mismatches,^{9,11,13,14,15} since 3′ end mismatches can disrupt the nearby polymerase active site.^17,18Strategies to alter mismatch impact, eg, degenerate/modified bases or extensive adaptation of PCR conditions, can prove helpful in specific situations, but these strategies often require a lot of time-consuming optimization and can result in unwanted side effects (eg, increased primer-dimer formation). Quantitative data on the effects of 3′ end mismatches is necessary to improve knowledge and reliable prediction of mismatch behavior, which is beneficial for the development and optimization of real-time PCR assays involving mismatches.Several studies on the effects of 3′ end primer-template mismatches have been published.^{9,10,19,20,21,22} However, only few systematically examined the behavior of 3′ end primer-template mismatches (including the relationship between these effects and the position of the mismatch) using modern quantitative methods. In this study, we comprehensively investigate the effects of 3′ end primer-template mismatches using different commercially available 5′-nuclease assay master mixes. Diagnostic laboratories often employ such optimized pre-mixed reagents, which are generally directly used with few adaptations. Our approach therefore provides a relevant system for quantification of mismatch impact on diagnostic real-time PCR assays. Our experiments resulted in a large quantitative dataset from which different aspects of mismatch effects on PCR amplification were further analyzed, ultimately leading to the formulation of a set of general guidelines for improved prediction of primer-template mismatch impact.     

High-Throughput Identification and Quantification of Candida Species Using High Resolution Derivative Melt Analysis of Panfungal Amplicons

Fungal infections pose unique challenges to molecular diagnostics; fungal molecular diagnostics consequently lags behind bacterial and viral counterparts. Nevertheless, fungal infections are often life-threatening, and early detection and identification of species is crucial to successful intervention. A high throughput PCR-based method is needed that is independent of culture, is sensitive to the level of one fungal cell per milliliter of blood or other tissue types, and is capable of detecting species and resistance mutations. We introduce the use of high resolution melt analysis, in combination with more sensitive, inclusive, and appropriately positioned panfungal primers, to address these needs. PCR-based amplification of the variable internal transcribed regions of the rDNA genes generates an amplicon whose sequence melts with a shape that is characteristic and therefore diagnostic of the species. Simple analysis of the differences between test and reference melt curves generates a single number that calls the species. Early indications suggest that high resolution melt analysis can distinguish all eight major species of Candida of clinical significance without interference from excess human DNA. Candida species, including mixed and novel species, can be identified directly in vaginal samples. This tool can potentially detect, count, and identify fungi in hundreds of samples per day without further manipulation, costs, or delays, offering a major step forward in fungal molecular diagnostics.Rapid and economical detection, identification, and quantification of fungal species directly from clinical samples is a long-sought goal of clinicians that has still not been fulfilled.^1,2,3,4,5,6 Culture-based diagnosis of fungal infections is inadequate in that many species do not culture efficiently or require unacceptably long incubations.⁷ Antigen-based tests for galactomannan or β-glucan are improvements over culture, but are either too specific, too insensitive, plagued by false positives, or not yet validated by widespread testing.^{8,9,10,11,12,13} Identification of C. albicans and C. glabrata by Peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) is in clinical use. However, this tool requires an initial culture step to increase fungal titer to detectable levels and is limited in the number of species it can identify.^14,15,16,17PCR-based strategies are the most likely solutions to challenges posed by fungal diagnostics. However, clinical diagnosis of fungal infections by PCR is perhaps its most challenging application, due to low cell numbers, potentially <1 cell/ml sample, to the added problems in lysing fungal walls, and to the similarity in rDNA sequences to human. It is clear that PCR is sufficiently sensitive and specific by in vitro testing, but sample processing under these extreme demands remains problematic. Reviews from 2002 to 2008 indicate that both the promise and problems are great.^6,8,18,19 Most approaches detect positives in clinical samples at their limits of detection, meaning they lack the level of robustness needed to avoid false negatives when widely applied.^1,20PCR strategies using panfungal primers that complement conserved regions of rDNA but span the variable internal transcribed spacer regions (ITS1 and ITS2) have the strong advantage that any and all fungal species will be captured in a single reaction.^{21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47} Traditionally, these amplicons are then sequenced to identify species, using standard, automated capillary sequencing, pyrosequencing, or sequencing-grade microarrays.^{27, 29, 32,33,34,35, 37, 44, 47,48,49,50} Alternatively, precise determination of the base composition of the amplicons by electrospray mass spectroscopy may identify species.⁵¹ Less precise but adequate resolution may be achieved by restriction enzyme analysis of the amplicon.^24,30 Repetitive sequence-PCR (REP-PCR), a version of randomly amplified polymorphic DNA (RAPD) in which primers target repetitive sequence elements, have been used for fungal identification.^52,53 However, this requires pure cultures as the starting material, which is useful in some applications but is not an acceptable precondition for a clinical fungal diagnostic tool. An alternative is to identify species with probes, either standard hybridization after PCR, or during amplification using Taqman, Beacon, or Scorpion probes,^{22,25,28,38,41,45} or hybridization-based fluorescence resonance energy transfer (FRET) probes.⁵⁴An alternative is the use of species-specific PCR, which is typically more sensitive and does not require sequencing of product. Species that are certain to be seen with reasonable frequencies can be detected by species-specific PCR. Approximately 80% of these are species of Candida (C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, and C. lusitaniae), or Aspergillus (A. fumigatus, A. flavus, A. terreus, A. niger). The remaining ∼20% include Fusarium, Sporothrix schenckii, zygomycetes (Absidia corymbifera, Rhizomucor pusillus, Rhizopus arrhizus, Mucor, and Cunninghamella). Some of the less common species are also the most problematic in terms of resistance or virulence. There are a number of publications reporting a variety of primers for this approach, with widely varying levels of rigor in their validation.^{55,56,57,58,59,60,61,62} In general, this approach has the disadvantage that multiple assays have to be run on each sample, adding cost and labor. Multiplexing is a possible alternative, but this is widely associated with reduced sensitivity. A further limitation is that many clinical samples will have novel species that may be missed by these primers.High resolution melt analysis is likely to provide an even simpler, faster, and cheaper identification tool sufficiently specific for fungal speciation. This approach more fully exploits the shape of the melting curve of an amplicon, which is a much richer source of information than melting temperature alone. Short, regional sequences denature to form single stranded regions, which release double-stranded DNA-binding fluorescent dyes, before reaching the temperature at which the entire amplicon denatures. This influences the shape of the melt curve, to generate nuances that reflect species-specific sequence differences. Resolution can be further enhanced or normalized by several methods.^63,64 This has enabled identification of bacterial and viral species.⁶⁵Our application of this tool to species of Candida shows that the separation between species is great enough to call species without any postamplification handling.     

Eliminating Animal Facility Light-at-Night Contamination and Its Effect on Circadian Regulation of Rodent Physiology, Tumor Growth, and Metabolism: A Challenge in the Relocation of a Cancer Research Laboratory

Appropriate laboratory animal facility lighting and lighting protocols are essential for maintaining the health and wellbeing of laboratory animals and ensuring the credible outcome of scientific investigations. Our recent experience in relocating to a new laboratory facility illustrates the importance of these considerations. Previous studies in our laboratory demonstrated that animal room contamination with light-at-night (LAN) of as little as 0.2 lx at rodent eye level during an otherwise normal dark-phase disrupted host circadian rhythms and stimulated the metabolism and proliferation of human cancer xenografts in rats. Here we examined how simple improvements in facility design at our new location completely eliminated dark-phase LAN contamination and restored normal circadian rhythms in nontumor-bearing rats and normal tumor metabolism and growth in host rats bearing tissue-isolated MCF7(SR^–) human breast tumor xenografts or 7288CTC rodent hepatomas. Reducing LAN contamination in the animal quarters from 24.5 ± 2.5 lx to nondetectable levels (complete darkness) restored normal circadian regulation of rodent arterial blood melatonin, glucose, total fatty and linoleic acid concentrations, tumor uptake of O₂, glucose, total fatty acid and CO₂ production and tumor levels of cAMP, triglycerides, free fatty acids, phospholipids, and cholesterol esters, as well as extracellular-signal-regulated kinase, mitogen-activated protein kinase, serine–threonine protein kinase, glycogen synthase kinase 3β, γ-histone 2AX, and proliferating cell nuclear antigen.Abbreviation: 13-HODE, 13-hydroxyoctadecadienoic acid; γH2AX, histone 2AX; AKT, serine–threonine protein kinase; ERK1/2, extracellular signal-regulated kinase p44/46; GSK3β, glycogen synthase kinase 3β: LAN light at night; MEK, mitogen-activated protein kinase kinase, PCNA, proliferating cell nuclear antigen; SR^–, steroid-receptor–negativeRelocating laboratory animal research from one institution to another can be a daunting task for both scientists and animal care personnel with regard to control of lighting and elimination of light-at-night (LAN) contamination. Appropriate facility lighting and lighting protocols, as outlined in the Guide for the Care and Use of Laboratory Animals,³⁰ are essential for maintaining the health and wellbeing of laboratory animals and ensuring the credible outcome of scientific investigations.^16-18,22 The profound effect of light on circadian behavior and physiology is well established.^{2,3,5,9,11,12,16-18,22,29,32,44,46,49,52,55-58,64}Minor alterations in light intensity,¹¹ spectral quality,¹² and duration⁹ at any given time of day can alter or disrupt chronobiologic rhythms markedly in all mammals.^{6,17,26,44,55-59} Light information, which initially is detected by a small group of intrinsically photosensitive retinal ganglion cells containing the blue light-sensitive photopigment melanopsin,^6,26 is transmitted through the retinohypothalamic tract⁵⁹ to a central molecular clock located in the suprachiasmatic nucleus of the hypothalamus.^32,57 The suprachiasmatic nucleus, the activity of which is entrained by the light:dark cycle,^32,57 sends projections over a polysynaptic pathway to the pineal gland driving a series of molecular events leading to the production of the pineal neurohormone melatonin (N-acetyl-5 methoxytryptamine), primarily during the night.^29,46 The daily rhythmic melatonin signal contributes to the temporal coordination of normal behavioral and physiologic functions including the sleep–wake^23,46,66 and reproductive cycles,^51,55 immune function,^38,41,56 hormone levels,^{19,31,45,47,68} temperature regulation,²³ electrolyte balance,⁶⁹ neural protein synthesis,⁶⁵ and redox states.^24,53Dark-phase LAN exposure suppresses endogenous melatonin concentrations and may lead to various disease states,^42,58 including carcinogenesis,^7,8,16,18,33 and metabolic syndrome.^{17,34-37,39,70} Previous in vivo studies in our former laboratory (at the Bassett Research Institute, Cooperstown, NY) demonstrated that animal room LAN of as little as 0.2 lx (0.08 µW/cm²; rodent eye level) during an otherwise normal dark-phase suppressed normal physiologic nighttime melatonin levels, leading to markedly disrupted circadian regulation of physiology and metabolism in nontumor-bearing host animals^16,18 and a stimulation in metabolism and proliferation of both tissue-isolated MCF7 steroid-receptor–negative (SR^–) human breast cancer xenografts and syngeneic grafts of rodent hepatoma 7288CTC in rats.^7,17 This effect was mediated by melatonin receptor-mediated suppression of cAMP, leading to inhibition of tumor linoleic acid uptake and its metabolism to the mitogenic signaling molecule 13-hydroxyoctadecadienoic acid (13-HODE). These events culminated in downregulation of epidermal growth factor and insulin-like growth factor 1 pathways.^7,8,16-19,62Exposure to LAN is likely to exert pervasive and problematic effects on mammalian behavior and physiology in laboratory animal facilities around the world. During the past decade, improved facility design and better adherence to animal room lighting protocols certainly has helped to reduce the problem. In moving to our laboratory animal facility at Tulane University School of Medicine (New Orleans, LA), we discovered considerable preexisting LAN contamination that had to be eliminated before we could resume our human cancer research.The current study was performed to monitor the effects of the elimination of animal room LAN contamination over time on animal health and wellbeing, tumor growth, and metabolic profiles by assessing well-established circadian parameters in physiology and metabolism.^7,8,16-18 We measured light-induced suppression of melatonin, an accepted and sensitive marker of the effects of light on the circadian system in all mammals,^{2,3,5,9,11,12,15,16,18,20,21,29,44,46,49,52,55-58,64} before and after tumor implantation and growth. Tissue-isolated MCF7(SR^–) human breast cancer xenografts and 7288CTC rat hepatomas have been well-characterized over the years in our former light-tight facilities^7,8,16-18 and provided us with unique markers and measures of the extent to which LAN contaminated our new animal quarters. In tumor-bearing animals exposed to even minimal LAN, the latency-to-onset of tumor growth and proliferation rates of these tumors increase markedly in direct proportion to LAN intensity. As improvements were made to eliminate LAN contamination in the new location over the course of more than 20 generations of tumor passages, we measured the changes and reestablishment of normal rat and host–tumor circadian regulation. The information from this study may assist investigators and animal care personnel in addressing this important influence on the health and wellbeing of laboratory animals and consequent effects on the outcome of scientific investigations.     

Progression of Ulcerative Dermatitis Lesions in C57BL/6Crl Mice and the Development of a Scoring System for Dermatitis Lesions

Ulcerative dermatitis (UD) is a common, spontaneous condition in mice with a C57BL/6 background. Although initial lesions may be mild, UD is a progressive disease that often results in ulcerations or debilitating fibrotic contractures. In addition, lesions typically are unresponsive to treatment. Euthanasia is often warranted in severe cases, thereby affecting study outcomes through the loss of research subjects. Because the clinical assessment of UD can be subjective, a quantitative scoring method and documentation of the likely time-frame of progression may be helpful in predicting when animals that develop dermatitis should be removed from a study. Such a system may also be helpful in quantitatively assessing success of various treatment strategies and be valuable to clinical laboratory animal veterinarians. In this 1.5-y, prospective cohort study, we followed 200 mice to monitor the development and course of UD. Mice were examined every 2 wk. A clinical sign (alopecia, pruritus, or peripheral lymphadenopathy) was not identified that predicted development of UD lesions in the subsequent 2-wk period. Once UD developed, pruritus, the character of the lesion (single or multiple crust, coalescing crust, erosion, or ulceration), and the size of the lesion were the only parameters that changed (increased) over the course of the disease. Pruritus was a factor in the rapid progression of UD lesions. We used these findings to develop a quantitative scoring system for the severity of UD. This enhanced understanding of the progression of UD and the quantitative scoring system will enhance the monitoring of UD.Abbreviation: UD, ulcerative dermatitis; S number, scratching number; COL, character of lesionsUlcerative dermatitis (UD) is an idiopathic, spontaneous, debilitating syndrome of laboratory mice that is typically a disease of aged^1,19,43,46 C57BL/6 mice or genetically engineered mice on a C57BL/6 background.^1,19,43,44 Some reports discuss a similar condition in young, weanling mice that presents initially as alopecia.^24,42,44,45 Prevalence rates of UD between 4.1% to 21% have been reported.^1,6,19 Although no etiology has been identified, environmental factors,^{6,19,41,42,44} diet,^{5,29,41,42,46} season,^19,41,43,44 age at weaning,⁴² alopecia,^24,42,44,45 sex,^19,39,41,43 immune complex vasculitis,¹ follicular dysplasia,⁴⁴ lesion location,²⁰ and deficiencies in vitamin A metabolism⁴⁴ have all been implicated as predisposing factors for disease development. In addition, oronasal pain and chronic inflammation may lead to self-mutilation as a result of, initiating an “itch” response.¹⁰ UD is diagnosed by ruling out other causes of dermatitis in laboratory mice, such as fur mites,⁹ infections, fight wounds,¹⁷ strain phenotype,^15,35,40,49 and experimentally induced dermatitis.^4,50 Other diagnostic criteria are based on professional judgment and may include strain (C57BL/6 background),^1,19,44 lesion location (head and dorsal thorax),^1,19,43,44 intense pruritus,^1,19,44 peripheral lymphadenopathy,^6,19,39 and failure to respond to treatment.¹⁹ The rapid progression of UD lesions results in significant morbidity in laboratory mice.^6,19,44 Typically, the lesions progress to large, irregularly shaped, confluent ulcerations on the dorsal cervical and thoracic region.^1,19,39,44 As the lesions heal, contracted scar tissue forms, which can impair species-typical behaviors and mobility.^39,41,43,44 The presence of large dermal ulcerations or debilitating contractures affect animal welfare and typically necessitate euthanasia of affected mice. Although reports on the later stages of UD have been consistent,^{1,19,39,41,43,44} information on the initiation and progression of UD lesions is conflicting. Pruritus,^1,19,44 pain,¹⁰ and genetic predisposition^1,19,43,44 have been implicated as initiators of the disease. Alopecia, pruritus, erythema, and single or multiple(s) crust have all been reported as early signs of the disease.^{1,19,39,42-44} However, the majority of this information has been collected retrospectively, at timed necropsies, or based on anecdotal reports.Scoring systems are useful tools to evaluate clinical diseases in laboratory animals. For example, scoring systems have been published for tumors,^14,28 body condition,^14,28,47 and neurologic phenotype¹³ in mice to aid in assessment of clinical disease severity. Even though the progressive and severe nature of UD typically warrants eventual euthanasia, determining the severity of disease has typically been based on professional judgment,^1,39 subjective scoring,^{12,15,19,40,43,48,49} or postmortem histology.^40,44 A quantitative scoring system for UD in live mice has not been described and could greatly aid laboratory animal veterinarians and researchers in determining the severity of the disease and response to treatment.The purpose of the current study is to investigate clinical parameters that reflect the progression of UD to facilitate management and veterinary care of mice with UD. We followed 200 mice from 3 wk of age until the development of UD to determine the initial signs and progression of UD lesions. We hypothesized that mice will first develop signs of pruritus prior to any clinical lesion. From there, we predicted that clinical lesions will progress stepwise though the following stages of severity, with or without alopecia: (1) excoriations; (2) a single, small punctuate crust; (3) multiple, small punctuate crusts; (4) coalescing crust; (5) erosion; and (6) ulceration. In addition, we hypothesized that a quantitative, validated, and reliable UD scoring system can be created that is based on physical examination parameters that do, in fact, predict development and progression of UD. Having a more thorough understanding of the initiation and progression of ulcerative lesions likely will enhance our ability to predict the outcome for a given mouse and develop earlier end-points for that mouse. Furthermore, use of this scoring system will enable accurate monitoring of UD lesions.     

A Simple, High-Throughput Assay for Fragile X Expanded Alleles Using Triple Repeat Primed PCR and Capillary Electrophoresis

Population screening has been proposed for Fragile X syndrome to identify premutation carrier females and affected newborns. We developed a PCR-based assay capable of quickly detecting the presence or absence of an expanded FMR1 allele with high sensitivity and specificity. This assay combines a triplet repeat primed PCR with high-throughput automated capillary electrophoresis. We evaluated assay performance using archived samples sent for Fragile X diagnostic testing representing a range of Fragile X CGG-repeat expansions. Two hundred five previously genotyped samples were tested with the new assay. Data were analyzed for the presence of a trinucleotide “ladder” extending beyond 55 repeats, which was set as a cut-off to identify expanded FMR1 alleles. We identified expanded FMR1 alleles in 132 samples (59 premutation, 71 full mutation, 2 mosaics) and normal FMR1 alleles in 73 samples. We found 100% concordance with previous results from PCR and Southern blot analyses. In addition, we show feasibility of using this assay with DNA extracted from dried-blood spots. Using a single PCR combined with high-throughput fragment analysis on the automated capillary electrophoresis instrument, we developed a rapid and reproducible PCR-based laboratory assay that meets many of the requirements for a first-tier test for population screening.Expansions of polymorphic (CGG)_n repeats beyond the normal range in the 5′ untranslated region of the Fragile X Mental Retardation (FMR1) gene on the X chromosome give rise to a variety of developmental and degenerative disorders, such as Fragile X syndrome (FXS), autism, Fragile X-associated tremor/ataxia syndrome (FXTAS) and primary ovarian insufficiency.^1,2,3 FXS is the most common inherited form of mental retardation and related intellectual and developmental disabilities in affected individuals.¹ In FXS patients, CGG repeats expanded beyond 200 (full mutation) become abnormally methylated.^2,3 Hypermethylation of the CpG islands in the 5′-untranslated region of the FMR1 gene in the full-mutation individuals leads to FMR1 gene silencing and complete absence of the gene product FMRP protein.³ FMRP is an RNA-binding protein, which has been shown to play an important role in mRNA translation, dendritic transport of mRNAs, and protein-synthesis dependent synaptic plasticity.^2,3,4The frequency of full mutations in the general population is thought to be approximately 1 in 4000 males and 1 in 8000 females.^1,5,6,7 Higher full-mutation allele frequency (1 in 2500 in females) has been documented in some countries.^{8,9,10,11,12,13}Alleles between 55 and 200 CGG repeats are classified as premutations. CpG islands in premutation FMR1 alleles are not methylated, and carriers of premutation alleles generally have normal levels of FMRP; however, CGG repeats in premutation alleles are unstable and may increase in size from generation to generation by maternal transmission, ultimately resulting in an affected newborn with a fully expanded allele. In fact, CGG expansion from 56 repeats in the mother to full mutation (538 repeats) in a child was reported.⁹ Recent studies have estimated a premutation allele frequency of 1 in 113–259 females and 1 in 260–800 males.^5,10,11 It has been described that premutation allele carriers have higher prevalence of learning disabilities, ADHD, and autism than carriers of non-expanded alleles.^12,13 Premutation females have higher rates of primary ovarian insufficiency: 20% in carriers versus 1% in normals.¹⁴ The size of the premutation may be a contributing factor to this condition.¹⁵ Furthermore, Fragile X-associated tremor/ataxia syndrome (FXTAS), a neurological disorder with symptoms including intention tremor, cerebellar ataxia, memory loss, dementia, and anxiety affects 20–40% premutation male and 8% of premutation female carriers who are over the age of 50.^16,17Molecular testing to diagnose Fragile X syndrome or identify premutation allele carriers has been available for over a decade. Methodologies for FXS diagnostic testing include Southern blot of the genomic DNA and PCR amplification of CGG repeat regions. Methylation-specific PCR can identify males with full mutations. Antibody-based detection of FMRP expression, and recently a mass spectrometry–based assay for FMRP^18,19 are less common diagnostic methods. Each method has certain advantages and disadvantages. For example, Southern analysis can detect full mutation, methylation and large premutations, but it cannot detect small premutations. An antibody test for FMRP can be used for affected males but cannot identify female carriers or distinguish between normal and premutation carriers.²⁰PCR amplification of the CGG repeat region in FMR1 in combination with Southern blot are the most commonly used techniques for CGG repeat detection and sizing.^21,22 PCR can easily detect normal alleles and most premutations; however, amplification of large premutations and full mutations is technically challenging. This is particularly difficult when working with specimens from females. An apparently normal homozygous result does not rule out the possibility of preferential amplification of the normal allele in the presence of full mutation that was not amplified by PCR.Population screening has been proposed to identify premutation allele carrier females and affected newborns.^{7,23,24,25,26} To make screening viable, a simple, inexpensive test with high sensitivity and specificity is necessary. We previously reported a simple triplet repeat primed PCR (TRP PCR) based assay that amplifies CGG repeats in the FMR1 gene and allows rapid detection of the “stutters” formed by the chimeric primer which binds inside the CGG repeat region.²⁷ Briefly, in TRP PCR, one primer is anchored completely outside of the CGG repeat region, while the other overlaps the CGG repeat and the adjacent unique sequence. Multiple amplicons are produced, each with a length difference of 3 bases. The “stutter” or ‘ladder’ produced on electrophoresis is the basis of this assay. Similar approaches have been reported by other groups using ethidium-stained agarose gels to detect “smears” formed by the chimeric primer.²⁸ In an effort to develop a high-throughput research Fragile X assay capable of identifying the presence or absence of an expanded FMR1 allele in a sample, we combined a TRP PCR^28,29,30 using a fluorescently labeled primer with fragment analysis on an Applied Biosystems PRISM 3100 genetic analyzer (3100). In this study we evaluated the assay using archived samples from routine clinical practice representing a range of FMR1 CGG repeats from normal through fully expanded alleles. We also show feasibility of running the TRP PCR assay with dried blood spot (DBS) samples.     

Multiple Extrauterine Pregnancy with Early and Near Full-Term Mummified Fetuses in a New Zealand White Rabbit (Oryctolagus cuniculus)

Extrauterine pregnancy (EP) is infrequent in mammalian species and occurs when fertilized ova implant and develop outside the uterus. A common outcome is abdominal pregnancy resulting in mummified fetuses (lithopedia). Here we describe an unusual case of abdominal pregnancy with early and near full-term lithopedia. Macroscopic findings supported the diagnosis of lithopedia with distinct age differences and facilitated further characterization of primary ectopia and risk factors leading to this occurrence.Abbreviation: EP, extrauterine pregnancyExtrauterine pregnancy (EP) occurs infrequently in most mammalian species.¹² The term derives from the Latin prefix meaning ‘outside’ or ‘beyond’ and refers to the implantation of a fertilized ovum outside the uterine cavity. Extrauterine pregnancy was first recognized more than 900 y ago² among other discoveries with a hereditary nature.¹³ Early reports compared EP in women, cats, dogs, and rabbits⁷ and described the presence of mummified fetuses in laboratory rabbits.^16,35EP is a serious obstetric complication that occurs asymptomatically in most cases.¹⁷ There are 4 classifications of EP: tubal, ovarian, abdominal–peritoneal, and cervical. The fallopian tube is the most common location and leads to tubal pregnancy. When gestation occurs in the abdominal–peritoneal cavity, abdominal pregnancy results and is subdivided as primary, when fertilization occurs outside the uterus after an oocyte is accidentally released from the fimbria, and secondary, when an oocyte is released due to direct tubal trauma.⁴⁵ A rare form of EP associated with high maternal morbidity and fetal mortality is called heterotopic (or combined) pregnancy, which occurs when 2 fertilized eggs coexist, one outside the uterus and the other inside.^18,33,44Undetected EP is frequently associated with fatal outcomes to the dam and offspring, including the formation of mummified fetuses, which may eventually become calcified and are called lithopedia (from the Greek: lithos, stone; paidion, child).^11,51,53 The condition is infrequent, and the factors that influence the unexpected outcomes of this pathology are not well understood.^26,34,35 Epizootiologic investigations are few,¹² although a recent report outlined the prevalence of EP in large NZW rabbit breeding colonies.⁵¹Examples of EP have been documented in dogs,¹⁷ cats,^14,39,42,49 rabbits,^20,29,45,51 hamsters,^9,46 rats,²⁶ mice,^8,12 guinea pigs,^3,30 lambs,⁴⁰ nonhuman primates,^10,34,38,50 and other species including humans.^11,12 However, despite the number of documented species, the majority of reports failed to note detailed clinical symptoms that interfered with reproduction even in instances that led to the formation of lithopedions.⁴²Experimentally, mouse embryos have successfully been transferred to a variety of sites including the peritoneal cavity, kidney, spleen, muscles, testis, and the anterior chamber of the eye.^1,6,21,31 The aim of the current report is to describe a rare case of abdominal pregnancy in which lithopedia developed clinically silently and coexisted with multiple pregnancies in a healthy doe rabbit.     

Comparison of the Effects of Ketamine, Ketamine�CMedetomidine, and Ketamine�CMidazolam on Physiologic Parameters and Anesthesia-Induced Stress in Rhesus (Macaca mulatta) and Cynomolgus (Macaca fascicularis) Macaques

This study compared the cardiovascular, respiratory, anesthetic, and glucocorticoid effects of ketamine alone with ketamine–medetomidine and ketamine–midazolam in rhesus and cynomolgus macaques. Macaques were given either intramuscular ketamine (10 mg/kg), intramuscular ketamine–medetomidine (3 mg/kg; 0.15 mg/kg), or oral midazolam (1 mg/kg) followed by intramuscular ketamine (8 mg/kg). The addition of medetomidine, but not midazolam, provided muscle relaxation and abolishment of reflexes that was superior to ketamine alone. In addition, medetomidine did not cause clinically relevant effects on cardiovascular and respiratory parameters when compared with ketamine. These 3 protocols did not have significantly different effects on fecal glucocorticoid metabolites. These results suggest that medetomidine can be a valuable addition to ketamine for healthy patients, whereas oral midazolam at the tested dose does not provide additional benefits.Abbreviations: KetMed, ketamine–medetomidine, KetMid, ketamine–midazolam, NHP, nonhuman primates, PaCO₂, arterial partial pressure of carbon dioxide, PaO₂, arterial partial pressure of oxygen, SO₂, oxygen saturation of hemoglobin, SaO₂, arterial oxygen saturation, SpO₂, peripheral oxygen saturation, T, timeKetamine hydrochloride is commonly used as a sole anesthetic agent in nonhuman primates (NHP) but has several drawbacks that could be ameliorated by combining it with other agents. First, animals sedated with dissociative anesthetics retain their reflexes and can exhibit varying amounts of skeletal muscle movement and, rarely, seizure activity.⁴⁶ This propensity raises concerns about personnel safety when working with animals carrying zoonotic infectious agents. In addition, excessive movement creates challenges when performing certain tasks such as tuberculosis testing or when monitoring animals for appropriate anesthetic depth during minor surgical procedures.⁵⁵ Second, the analgesic properties of ketamine in NHP are unknown. Ketamine generally is considered to be satisfactory for somatic analgesia but inadequate for deep or visceral pain. However, analgesic effects can differ widely between species, and the few studies available show variable results even for minor procedures in NHP.^21,28,53 In addition, ketamine is associated with pain on injection and volume-dependent tissue damage in many species^{7,17,18,46,55} and can cause severe psychomimetic side effects such as hallucinations and delirium in people.^29,44 Although we cannot know whether ketamine causes excessive psychological stress in NHP, policies of the Public Health Service and Animal and Plant Health Inspection Services establish a precedent for assuming that procedures affect animals as they do human beings unless there is evidence to the contrary.^3,43Medetomidine and midazolam are 2 agents that are often combined with ketamine to improve anesthesia.^{15,24,27,33,40,49,50,54,58,59} Medetomidine is a selective α2-adrenergic agonist that is a racemic mixture of 2 optical enantiomers, the active dexmedetomidine and the inactive levomedetomidine. Although the primary benefit of α2 agonists is improved muscle relaxation, they also provide documented analgesic effects in many species, including humans and NHP.^{10,11,23,35,46,57} In addition, reversal of the medetomidine with atipamezole can decrease recovery time and reverse sedation if unacceptable side effects occur.⁵⁵ As a benzodiazepine, midazolam can be expected to offer additional sedation and muscle relaxation to ketamine anesthesia.⁴⁵ In addition, the use of medetomidine or midazolam may accommodate a decreased dose of ketamine, thus reducing tissue necrosis and inflammation.⁵⁵In addition to the physical benefits of medetomidine and midazolam, these drugs may help attenuate anesthesia-related stress responses. In dogs, medetomidine decreases the stress-related hormonal changes that occur with ketamine alone.¹ Human physicians often use benzodiazepines to decrease psychotic events, anxiety, and nausea, particularly when ketamine is used.^{8,9,19,22,29,44} In NHP, premedication with midazolam may decrease the stress associated with injection and the possible psychologic effects associated with ketamine. These potential benefits are difficult to measure in animals, but various methods have been used in an effort to quantify the stress associated with anesthesia in NHP.^2,14,51,63 Food intake and urinary cortisol are affected significantly by ketamine anesthesia in macaques.^14,51 Fecal cortisol is gaining popularity as a noninvasive way to evaluate the hormonal impact of a variety of situations, including restraint and sedation.^{20,38,41,48,56,61,63} For example, fecal cortisol increased significantly in chimpanzees 2 d after sedation.⁶³ Although cortisol was the first hormone measured in fecal samples, a fecal corticosterone assay has been developed that measures glucocorticoid metabolites in primate fecal samples with less variable results than those of fecal cortisol assays.^60,62 Although any anesthetic episode may create stress, whether particular drugs or injections in an unsedated animal are exacerbating factors is unclear. We therefore used the glucocorticoid assay to compare 3 different anesthesia protocols.The purpose of this study was to determine whether medetomidine or oral midazolam could provide a deeper level of anesthesia in macaques than ketamine alone with comparable safety. In addition, we sought to determine whether either of these medications, particularly premedication with midazolam, attenuated the glucocorticoid response associated with anesthesia.     

Assessment of Routine Procedure Effect on Breathing Parameters in Mice by Using Whole-Body Plethysmography

We used whole-body plethysmography to investigate the effect of restraint, ear marking, tail vein and retroorbital blood sampling, and tail clipping on respiration in Balb/c × TCR-HA^+/– F1 hybrid mice (F1h). Baseline values of breathing parameters were determined. During the experiment, mice experienced a procedure and then plethysmographic recordings were obtained immediately and at 4, 24, and 48 h afterward. Baseline breathing parameters showed significant differences between sexes. Restraint affected minute volume differently than did handling in male mice and to a lesser extent in female mice. Ear marking significantly changed minute volume compared with handling but not restraint in male mice and in the opposite manner in female mice. Tail vein blood sampling changed minute volume in a significant manner compared with restraint but not compared with handling in both sexes. Retroorbital blood sampling significantly changed minute volume compared with values for both handling and restraint in male mice but only compared with handling in female mice. Tail clipping modified minute volume significantly compared with handling in male mice and compared with restraint in both sexes. Analysis of data showed that routine procedures affect minute volume in mice depending on invasiveness of maneuver and in a sex-biased manner for as long as 24 h after the procedure. Our experiment shows that procedures performed on laboratory mice can change respiratory parameters and can be investigated by plethysmography.Abbreviation: F1h, Balb/c × TCR-HA^+/– F1 hybrid miceHandling, restraint, identification methods, and blood or tissue sampling are regarded as routine procedures in animal experiments. This aspect is particularly important when working with transgenic animals, where the need for genotyping demands the frequent use of these procedures, which can readily yield DNA samples.⁵ The effect of routine procedures on physiologic parameters varies depending on method, frequency, and duration, but these procedures generally are considered to be acute stressors.^2,5,26,27 A large body of research has focused on the effect of routine procedures on animals’ physiology. These effects were assessed by studying changes in behavior,^1,23,36,37 body weight,^29,37 food and water intake,^29,37 body temperature³⁵ and heart rate by radiotelemetry,^{5,10,19,26,27} corticosterone^{11,12,24,25,29} and glucose levels,³³ and blood variables.^29,36 Several differences in the response of laboratory animals to stress have been correlated to breed,^2,33 strain,^11,14,37 age,²⁴ and sex.^12,23,24 Some authors²⁴ have noted that female mice show a lower overall stress level due to the modest effect of social competition. Another study¹² showed a different, sex-specific response to hypoxic ventilation in rats, which was higher in male than female rats. In addition, these authors¹² suggested that female ovarian hormones are prime candidates for stress regulation.Whole-body plethysmography is a noninvasive, indirect method of studying respiratory function in conscious, unrestrained animals. This method has been used particularly in pharmacologic and toxicologic studies in diverse animal species including mice,^3,9,14,15,38 rats,^8,20 cats,²² dogs,^16,34 and pigs.¹³ The present study was designed to investigate the extent to which routine procedures affect breathing parameters in a transgenic mouse colony. Male and female mice were exposed to 4 routine procedures that are used for DNA sampling and identification of transgenic mice (ear marking, tail clipping, and tail vein and retroorbital blood sampling). Respiratory parameters were recorded by whole-body plethysmography immediately and at 4, 24, and 48 h after the procedure. Handling and restraint were used as control procedures.     

Evaluation of Second-Generation Sequencing of 19 Dilated Cardiomyopathy Genes for Clinical Applications

Medical sequencing for diseases with locus and allelic heterogeneities has been limited by the high cost and low throughput of traditional sequencing technologies. “Second-generation” sequencing (SGS) technologies allow the parallel processing of a large number of genes and, therefore, offer great promise for medical sequencing; however, their use in clinical laboratories is still in its infancy. Our laboratory offers clinical resequencing for dilated cardiomyopathy (DCM) using an array-based platform that interrogates 19 of more than 30 genes known to cause DCM. We explored both the feasibility and cost effectiveness of using PCR amplification followed by SGS technology for sequencing these 19 genes in a set of five samples enriched for known sequence alterations (109 unique substitutions and 27 insertions and deletions). While the analytical sensitivity for substitutions was comparable to that of the DCM array (98%), SGS technology performed better than the DCM array for insertions and deletions (90.6% versus 58%). Overall, SGS performed substantially better than did the current array-based testing platform; however, the operational cost and projected turnaround time do not meet our current standards. Therefore, efficient capture methods and/or sample pooling strategies that shorten the turnaround time and decrease reagent and labor costs are needed before implementing this platform into routine clinical applications.Genetic testing for disorders with locus and allelic heterogeneity has been a challenge due to the high cost of sequencing entire coding regions of numerous genes. Classically, medical sequencing has used capillary-based “Sanger” sequencing technology and this has remained the gold standard for three decades. However, this method is expensive and has low throughput. It was not until novel technology platforms emerged that comprehensive testing became within reach. One such technology is array-based sequencing, which drastically increased the number of genes that could be analyzed simultaneously.^{1,2,3,4,5,6,7} We previously developed an array-based resequencing test for dilated cardiomyopathy (DCM), that doubled the number of genes analyzed in parallel while reducing test cost and turnaround time.⁶ However, this technology has two major drawbacks, particularly in a clinical setting. First, resequencing arrays have a poor detection rate for insertions and deletions (in/dels).^8,9 Second, the somewhat static nature of the chip design makes it time-consuming and impractical to add new content, especially in a disease area like DCM where genes are being discovered at a rapid pace. As such, novel technologies are needed to provide comprehensive sequencing of all DCM genes with high analytical sensitivity and with the goal of further reducing the cost of diagnostic testing.Second-generation sequencing (SGS) technologies, commonly referred to as “next-generation sequencing,” are based on massive parallel sequencing of millions of DNA templates through cycles of enzymatic treatment and image-based data acquisition. Several platforms have been developed in the last few years based on different biochemistries and cluster generation.^10,11,12 The most commonly used platforms include the Illumina Genome Analyzer (GAII; Solexa Technology),¹³ Roche Applied Sciences (454 sequencing),¹⁴ and ABI-Applied Biosystems (SOLiD platform).¹⁵ These technologies have been adopted for a wide variety of research applications^10,11,16 and have now matured sufficiently to be considered as robust enough for clinical applications. For example, SGS has recently been applied to resequencing the NF1 locus as well as the mitochondrial and small-cell lung cancer genomes.^17,18,19 In addition, whole exome resequencing has been conducted to discover genes underlying rare monogenic diseases.^20,21,22,23The specific advantages offered by SGS are twofold: the low cost per base and the ability to sequence millions of reads in parallel, allowing for simultaneous analysis of a large number of genes. However, these are offset by two major disadvantages: shorter reads and reduced accuracy as compared to Sanger sequencing.¹¹ Despite their disadvantages, improvements in these technologies promise to meet the technical requirements and strict quality standards of clinical diagnostics including analytical sensitivity, reproducibility and cost effectiveness.Several methodological approaches to capturing target gene regions have evolved to complement the higher capacity and throughput of novel sequencing technologies. These methods involve constructing and enriching a DNA “library” and use both PCR and/or hybridization as the mode of target selection. The classic PCR approach to library generation requires amplification of target regions, pooling, concatenation, shearing and ligation of adaptors and sequencing primers. Secondary droplet-based microfluidic technologies have evolved to facilitate high-throughput PCR in picoliter droplets.²⁴ With hybridization-based methods, libraries are constructed by shearing total gDNA followed by adaptor ligation and hybridization to oligonucleotides that are complementary to the desired target. Hybridization can be performed either on a solid surface array, on a filter, or by hybridization in solution^21,25,26,27 A third general approach to target selection uses molecular inversion probes. Molecular inversion probes consist of two primers linked together by a backbone and, similar to PCR, bind to specific target DNA. This is followed by gap filling, ligation, and enrichment steps.^28,29Important considerations in choosing a method include total amount of starting DNA, the size of the target region and the types of sequence alteration under investigation. Technical parameters such as target specificity, uniformity, and completeness of coverage also vary with each methodology.¹² Although each method has advantages or disadvantages depending on the application, we selected PCR, a well-established and robust targeted amplification method for this study.Leveraging on our array-based resequencing test for DCM,⁶ we evaluated the suitability of targeted PCR followed by Illumina GAII resequencing for a clinical testing environment. We used a number of samples enriched for a large number of substitutions as well as insertions and deletions in DCM genes to assess the analytical performance parameters. We also evaluated the cost and turnaround time of such a test and compared it to our current, array-based resequencing test.     

Multiplex Fluorescent Immunoassay for Detection of Mice Infected with Lactate Dehydrogenase Elevating Virus

Commercially available diagnostic tools for the detection of lactate dehydrogenase elevating virus (LDV) infection have been restricted to measurement of serum lactate dehydrogenase (LDH) activity levels and detection of the viral genome by RT-PCR assays. Serologic diagnosis of LDV infection has not been widely adopted due to the belief that the formation of antigen–antibody complexes and B-cell polyclonal activation may confound interpretation of results. In the current study, we inoculated BALB/c, C57BL/6, and Swiss Webster mice with LDV to compare the diagnostic reliability of a commercially available multiplex fluorescent immunoassay for the detection of antiLDV antibodies with that of the LDH enzyme assay. The serologic assay was vastly more sensitive and specific than was the LDH enzyme assay. Moreover, the serologic assay detected antiviral antibodies throughout the 3-mo time course of this study. These results suggest that antigen–antibody complex formation and polyclonal B-cell activation had little effect on assay performance.Abbreviation: LDV, lactate dehydrogenase elevating virus; MFI, multiplex fluorescent immunoassayLactate dehydrogenase elevating virus (LDV) is an RNA virus within the family Arteriviridae that initially was identified as a transmissible contaminant of tumors in laboratory mice.⁴¹ Previous reports have demonstrated that after experimental infection, circulating virus levels peak by 12 to 24 h, stabilize by 1 to 2 wk after infection, and persist for the lifetime of the mouse.^36,38-40 Although clinical disease is rare and only seen in immunocompromised strains, viral infection has many implications for biomedical research, including changes in tumor growth rate,^3,26,31,49 differences in immunologic function,^{21,29,32,33,35,44,45} polyclonal B-cell activation,¹² and altered serum enzyme levels.^34,39Laboratory mouse colonies are commonly screened for viral pathogens by serology, but commercial diagnostic methods for LDV have been limited to RT-PCR assays^10,20,47,48 and measurement of serum lactate dehydrogenase (LDH) activity. Although RT-PCR assays are a very reliable diagnostic method, it is costly and time intensive. Historically, measurement of serum or plasma LDH activity has been used to detect infection, but at least one study has determined that this assay has poor sensitivity and specificity.⁴⁸ LDV infection causes a 5- to 10-fold elevation of LDH within 3 d of exposure, and, due to decreased clearance rates, LDH levels can remain elevated for as long as 10 mo after exposure.¹ Enzyme activity is assayed by the coupled reaction catalyzed by lactate dehydrogenase:LDH activity (in units) is quantified as the change in optical density due to the oxidation of NADH or reduction of NAD⁺.¹⁸ When using LDH activity levels for diagnostic purposes, false positives may result from cellular damage and leakage of LDH due to repeated venipuncture¹⁶ or hemolysis.⁵⁰ Differences in baseline plasma levels of LDH between sexes and ages of inbred mice may confound the interpretation of results also.¹⁹ One published diagnostic method, not currently commercially available, is the detection of viral particles in plasma by using a latex agglutination assay.²⁹ However, this assay was used to detect acute LDV infection, and its utility for detecting chronic infection is unknown. In light of the weaknesses of the RT-PCR and LDH activity assays and the unavailability of the latex agglutination assay, we reexamined the suitability of a serologic assay for the detection of LDV.Serologic diagnosis of LDV infection has not received wide acceptance due to potentially confounding polyclonal B-cell activation and antigen–antibody complex formation.^17,37 Although polyclonal B-cell activation and antigen–antibody complex formation in LDV-infected mice are well described in the literature,^12,13,35 their effect on antiLDV antibody detection is unclear. Immunocompetent mice mount a robust immune response, but only a small fraction of the antibodies formed are specific for LDV proteins.¹⁵ AntiLDV antibodies are directed toward 2 of the 3 structural proteins: the 14-kDa nucleocapsid protein and the 24- to 42-kDa glycosylated envelope protein.^5,14 Questions about the sensitivity and specificity of antiLDV antibody detection arose when multiple studies demonstrated the presence of low-molecular–weight complexes in LDV immune serum that bound to ELISA plates not coated with antigen.^6,25 These complexes are believed to comprise auto-antibodies and cellular proteins rather than antiLDV antibodies and LDV viral proteins.^25,43 Application of a blocking agent to ELISA plates not coated with antigen reduced, but did not completely prevent, the binding of nonspecific antibodies to uncoated wells.²⁵ This result suggests that nonspecific antibodies may interfere with the detection and quantification of antiLDV antibodies. However, to our knowledge, there have been no controlled studies to show that these nonspecific antibodies compromise the sensitivity or specificity of antiLDV antibody detection in murine serum samples. Contrary to the suggested interference of these low-molecular–weight complexes, many reports have shown that LDV-infected mice mount a demonstrable antibody response, as detected by ELISA and immunofluorescent assays, as early as 1 wk after infection and which remains elevated for at least 1 y.^5,7,14,30,40 These methods are not routinely used in high-throughput screening laboratories because of the relative inefficiency of cultivating LDV: this virus does not replicate well in cell culture.⁴⁶The objective of the current study was to compare the diagnostic reliability of a commercially available multiplex fluorescent immunoassay (MFI) for the detection of antiLDV antibodies with that of the LDH activity assay in laboratory mice experimentally infected with LDV. To this end, we inoculated 2 inbred strains and a single outbred stock of laboratory mice with LDV or a sham inoculum and evaluated serum longitudinally over a 3-mo period for LDH enzyme activity, antiLDV antibodies by MFI, and LDV genomic DNA by RT-PCR assay. To confirm the presence or absence of viral genome in each mouse, spleen samples were evaluated by using an RT-PCR assay, the ‘gold standard.’ Our results demonstrate that the serologic diagnosis of LDV infection by using MFI was more sensitive and specific than was the LDH activity assay in female Swiss Webster, C57BL/6, and BALB/c mice at 2, 4, 8, and 12 wk after infection.     

Soiled Bedding Sentinels for the Detection of Fur Mites in Mice

Identification and eradication of murine fur mite infestations are ongoing challenges faced by many research institutions. Infestations with Myobia musculi and Myocoptes musculinus can lead to animal health problems and may impose unwanted research variables by affecting the immune and physiologic functions of mice. The purpose of this study was to evaluate the utility and efficacy of soiled bedding sentinels in the detection of fur mite infestations in colony mice. Female young-adult CRL:CD1(ICR) mice (n = 140) were exposed over a 12-wk period to various volume percentages of soiled bedding (11%, 20%, 50%, and 100%) from fur-mite–infested animals. Mice were tested every 2 wk with the cellophane tape test to identify the presence of fur mite adults and eggs. At the end of 12 wk, all mice exposed to 11%, 20%, and 50% soiled bedding tested negative for fur mites. One of the 35 mice (3%) receiving 100% soiled bedding tested positive for fur mites at the end of the 12-wk follow-up period. These findings suggest that the use of soiled bedding sentinels for the detection of fur mite infestations in colony mice is unreliable.Ectoparasite infestations present an ongoing threat to barrier facilities. Murine acariasis in laboratory mice frequently is caused by Myobia musculi, Myocoptes musculinus, and Radfordia affinis.^{1,13,17,40,41} These infestations can be challenging to identify and control and often lead to animal health problems and research complications. For this reason, many institutions strive to exclude these parasites from their barrier facilities.^1,17,18,41 Infestations can further compromise ongoing research by disrupting collaboration with institutions affected by sporadic or endemic mite infestations in their facilities.¹⁸Myocoptes musculinus is the most common fur mite identified among laboratory mice, although mixed infections with Myobia musculi are common.¹⁷ The life cycles of Myocoptes and Myobia are 14 and 23 d, respectively.^2,17 Myobia mites most frequently are found to inhabit the head and neck of mice, whereas Myocoptes are reported to have a predilection for the back, ventral abdomen, and inguinal regions.^2,17 Mite infestations in live animals are often diagnosed by using cellophane tape tests.^5,14,25 A clear piece of cellophane tape is pressed against the fur of the mouse, affixed to a slide, and examined microscopically for the presence of eggs or adult mites. Pelage collection and examination and skin scraping are 2 other common diagnostic methods. These tests have been shown to have increased sensitivity when compared with the tape test, but they have the disadvantage of requiring an anesthetized or recently euthanized animal.^2,5,17Fur mites feed on the superficial skin tissues and secretions of the animals they infest.^1,2,17 Mite infestations in mice have been associated with numerous health problems. Common clinical manifestations of acariasis include alopecia, pruritis, and scruffiness.^{1,2,10,15,17-20,22,26,31,42,44} Severe health problems including ulcerative dermatitis, hypersensitivity dermatitis, and pyoderma can develop also.^1,2,10,17,41 Infested mice may also be prone to secondary infections, reduced life span, and decreased body weight.^2,17,42 Several studies have analyzed the potential research complications associated with murine acariasis.^{10,15,18-20,22,26,31,42,44} Mite infestations have been shown to cause elevations in IgE, IgG, and IgA levels; mast cell degranulation; increased levels of inflammatory cytokines; and lymphocytopenia.^{18-20,22,26,31,44} The changes in the immunologic function of affected mice can persist even after mite eradication.¹⁸Multiple chemical treatment modalities have been proposed for the eradication of fur mites in infested animals.^{2,3,5,8,12,14,17,25,29,30,32,36,43} Conflicting information exists regarding the success of many of these treatment regimes. In addition, several of the proposed treatments have been associated with toxicity, adverse health effects in mice, and alterations in the physiologic or immune function of the animals.^{2,3,5,8,12,14,17,25,29,30,32,36,43} The complications associated with identifying an effective treatment for murine acariasis while minimizing toxicity and the introduction of unknown research variables highlight the importance of rapid and effective detection of mite infestations in barrier facilities.Many institutions rely on soiled bedding sentinels for their primary source of information on colony health status.^9,21,33,35 Several studies have demonstrated the efficacy of soiled bedding sentinels to detect common murine pathogens such as mouse hepatitis virus, mouse norovirus, Helicobacter spp., and pinworms.^{4,7,24,28,37,38} However, not all pathogens are easily transmitted through soiled bedding exposure. Agents that are not routinely identified through soiled bedding sentinels include those that are shed in low numbers, are susceptible to environmental factors, or are not easily transmitted through the fecal–oral route.^6,21,33 Examples of pathogens that are not easily transmitted or detected through soiled bedding exposure include mouse Sendai virus, Pasteurella pneumotropica, lymphocytic choriomeningitis virus, and cilia-associated respiratory bacillus.^7,9,11,16,35 In addition, the sensitivity of soiled bedding sentinel programs varies with the number of animals affected within the colony.^27,38In 2008, our institution faced a fur-mite outbreak that affected more than 25 rooms in a single barrier facility. Animals positive for Myobia musculi, Myocoptes musculinus, or both were identified through either health check requests for itching and scratching animals and by testing of animals scheduled for export to other institutions. Despite the extent of this outbreak, the soiled bedding sentinels in all mite-positive rooms consistently tested negative on cellophane tape tests for fur mites.To our knowledge, only one study has specifically examined the efficacy of soiled bedding sentinels in the detection of fur mites in mice.³⁴ A separate study, examining the transmission of mouse hepatitis virus to soiled bedding sentinels,³⁸ demonstrated that 75% of cages (3 of 4) exposed to soiled bedding from colony animals tested positive for fur mites after 19 wk of exposure. That previous study used 8 cages of 12 mice each; 4 cages received soiled bedding from colony animals, whereas the other 4 cages received clean nonsoiled bedding. In that study,³⁸ 56.3% of colony mice were known to be mite-positive. Other literature suggests that spread of mites to naïve animals requires direct contact and that soiled bedding does not serve as an effective mechanism for transmission.^1,17,23,39 However, we were unable to identify any research or experiments that substantiated these conclusions.The purpose of the present study was to evaluate whether CRL:CD1(ICR) mice housed in static microisolation caging on soiled bedding from mice with Myobia and Myocoptes infestations can be used as sentinels for the detection of fur mites and to determine how the efficacy of these soiled bedding sentinels for fur-mite detection varies with the prevalence of fur-mite infestation among colony animals.     

Assessment of Immune Activation in Mice before and after Eradication of Mite Infestation

Mite infestation of mice remains a persistent problem for many institutions, leading to numerous health problems and creating unknown and unwanted variables for research. In this study, mice with mite infestation demonstrated significantly higher levels of inflammatory cytokines, both at draining lymph nodes (axillary) and systemically, as compared with mice without mites. In addition, histologic evaluation revealed significant inflammation in mite-infested mice. Inflammatory changes were still present in the skin of mice at 6 to 8 wk after treatment, despite absence of detectable infestation at that time. Because these significant and lasting local and systemic changes have the potential to alter research findings, eradication of mites infestations should be an important goal for all institutions.Abbreviation: KC, keratinocyte-derived chemokine; MIP, macrophage inflammatory proteinLaboratory mice can harbor several species of acarids (fur mites), including Myobia musculi, Radfordia affinis, Myocoptes musculinus, and Psorergates simplex.^{11,14,29,40,45} Fur mites are an excluded pathogen in most research facilities, particularly within barrier suites, and in order to control or avoid mite infestations, many facilities, including those with ongoing infestations, will not accept infested animals from outside sources. Such policies can prevent or halt collaborative research between investigators in different institutions because mite infestation is a sporadic or endemic problem in many facilities that house mice under conventional conditions, despite attempts at eradication.^{12,22,25,43,62,69}Mite infestations cause several health problems in mice, including ulcerative dermatitis, amyloidosis, and other immune system alterations.^{2,12,22,27,29-31,37,44,45,61} For example, mite infestations are associated with increased serum concentrations of IgE and IgG in mice.^30,44,48 Alterations in immune responses could alter research data and thereby perhaps alter the associated conclusions.^36,65,66,70 Mice with mite infections often develop dermatitis, which can lead to bacterial infection and additional changes in immune status.^{15,30,31,45,61} Because any pathogenic infection can cause variability and alter basal measures of immune function, clinical chemistry, and behavior in mice, maintaining laboratory rodents in a disease-free state is crucial to their use for the collection of valid research data.⁵¹The eradication of external parasites is a difficult process. Many reports have been published that attempt mite eradication using various drug treatments,^{5-7,17,18,23,24,35,39,41-43,46,47,49,50,57,59,67} with each method having distinct advantages and disadvantages. Some, but not all, of these treatment regimens have been compared directly.¹⁰ The mite life cycle complicates treatment, because eggs and larvae can be less susceptible to drugs than are adult parasites.^2,19,20,55 In addition, mite eggs can contaminate the environment, providing a source for re-infection of treated animals.^20,63,64 Some drugs (for example, ivermectin) have been associated with toxicity and death in mice, especially among specific transgenic lines.^{8,12,28,53,55,69} Other drugs may require frequent or repeated treatment of the mice. Furthermore, the drugs themselves may have properties that alter physiology or immune function in animals.^2,13,60 The development of new veterinary drugs for treatment of parasites has increased the available therapies for rodent acariasis. Compounds such as fipronil and selamectin provide good efficacy against external parasites with limited side effects in mammals.^9,22,68Our facility housed a large colony of mice that occupied several rooms and were infested with Myocoptes musculinus and Myobia musculi. Although the majority of mite-infested mice had mild or no dermatitis, some infected mice had severe dermatitis. The goal of this study was to evaluate the local and systemic immune response in mice infested with mites. To our knowledge, this study is the first to comprehensively compare cytokine levels and histologic findings in mite-infested, treated, and mite-negative mice. We hypothesized that the immune response would be altered in mite-infested mice as demonstrated by significantly elevated cytokine levels in the draining lymph nodes or spleen as compared with mice that had never been infested with mites. In addition, we hypothesized that significant pathologic changes in the epidermis, dermis, and subcutaneous tissues would be present in response to mite infestation.     

Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

Nitric oxide (NO) is a ubiquitous mediator of inflammation and immunity, involved in the pathogenesis and control of infectious diseases, autoimmunity, and cancer. We observed that the expression of nitric oxide synthase-2 (NOS2/iNOS) positively correlates with Th17 responses in patients with ovarian cancer (OvCa). Although high concentrations of exogenous NO indiscriminately suppress the proliferation and differentiation of Th1, Th2, and Th17 cells, the physiological NO concentrations produced by patients’ myeloid-derived suppressor cells (MDSCs) support the development of RORγt(Rorc)⁺IL-23R⁺IL-17⁺ Th17 cells. Moreover, the development of Th17 cells from naive-, memory-, or tumor-infiltrating CD4⁺ T cells, driven by IL-1β/IL-6/IL-23/NO-producing MDSCs or by recombinant cytokines (IL-1β/IL-6/IL-23), is associated with the induction of endogenous NOS2 and NO production, and critically depends on NOS2 activity and the canonical cyclic guanosine monophosphate (cGMP)–cGMP-dependent protein kinase (cGK) pathway of NO signaling within CD4⁺ T cells. Inhibition of NOS2 or cGMP–cGK signaling abolishes the de novo induction of Th17 cells and selectively suppresses IL-17 production by established Th17 cells isolated from OvCa patients. Our data indicate that, apart from its previously recognized role as an effector mediator of Th17-associated inflammation, NO is also critically required for the induction and stability of human Th17 responses, providing new targets to manipulate Th17 responses in cancer, autoimmunity, and inflammatory diseases.Nitric oxide (NO; a product of nitrite reduction or the NO synthases NOS1, NOS2, and NOS3; Culotta and Koshland, 1992), is a pleiotropic regulator of neurotransmission, inflammation, and autoimmunity (Culotta and Koshland, 1992; Bogdan, 1998, 2001; Kolb and Kolb-Bachofen, 1998) implicated both in cancer progression and its immune-mediated elimination (Culotta and Koshland, 1992; Coussens and Werb, 2002; Hussain et al., 2003; Mantovani et al., 2008). In different mouse models, NO has been paradoxically shown to both promote inflammation (Farrell et al., 1992; Boughton-Smith et al., 1993; McCartney-Francis et al., 1993; Weinberg et al., 1994; Hooper et al., 1997) and to suppress autoimmune tissue damage through nonselective suppression of immune cell activation (Bogdan, 2001; Bogdan, 2011), especially at high concentrations (Mahidhara et al., 2003; Thomas et al., 2004; Niedbala et al., 2011). Although previous studies demonstrated a positive impact of NO on the induction of Th1 cells (Niedbala et al., 2002) and forkhead box P3–positive (FoxP3⁺) regulatory T (T reg) cells (Feng et al., 2008) in murine models, the regulation and function of the NO synthase (NOS)–NO system have shown profound differences between mice and humans (Schneemann and Schoedon, 2002, Schneemann and Schoedon, 2007; Fang, 2004), complicating the translation of these findings from mouse models to human disease.In cancer, NOS2-derived NO plays both cytotoxic and immunoregulatory functions (Bogdan, 2001). It can exert distinct effects on different subsets of tumor-infiltrating T cells (TILs), capable of blocking the development of cytotoxic T lymphocytes (CTLs; Bronte et al., 2003), suppressing Th1 and Th2 cytokine production, and modulating the development of FoxP3⁺ T reg cells (Brahmachari and Pahan, 2010; Lee et al., 2011). NOS2-driven NO production is a prominent feature of cancer-associated myeloid-derived suppressor cells (MDSCs; Mazzoni et al., 2002; Kusmartsev et al., 2004; Vuk-Pavlović et al., 2010; Bronte and Zanovello, 2005), which in the human system are characterized by a CD11b⁺CD33⁺HLA-DR^low/neg phenotype consisting of CD14⁺ monocytic (Serafini et al., 2006; Filipazzi et al., 2007; Hoechst et al., 2008; Obermajer et al., 2011) and CD15⁺ granulocytic (Zea et al., 2005; Mandruzzato et al., 2009; Rodriguez et al., 2009) subsets (Dolcetti et al., 2010; Nagaraj and Gabrilovich, 2010).Production of NO in chronic inflammation is supported by IFN-γ and IL-17 (Mazzoni et al., 2002; Miljkovic and Trajkovic, 2004), the cytokines produced by human Th17 cells (Veldhoen et al., 2006; Acosta-Rodriguez et al., 2007a,b; van Beelen et al., 2007; Wilson et al., 2007). Human Th17 cells secrete varying levels of IFN-γ (Acosta-Rodriguez et al., 2007a; Acosta-Rodriguez et al., 2007b; Kryczek et al., 2009; Miyahara et al., 2008; van Beelen et al., 2007; Wilson et al., 2007) and have been implicated both in tumor surveillance and tumor progression (Miyahara et al., 2008; Kryczek et al., 2009; Martin-Orozco and Dong, 2009). Induction of Th17 cells typically involves IL-1β, IL-6, and IL-23 (Bettelli et al., 2006; Acosta-Rodriguez et al., 2007a,b; Ivanov et al., 2006; van Beelen et al., 2007; Veldhoen et al., 2006; Wilson et al., 2007; Zhou et al., 2007), with the additional involvement of TGF-β in most mouse models (Bettelli et al., 2006; Mangan et al., 2006; Veldhoen et al., 2006; Zhou et al., 2007; Ghoreschi et al., 2010), but not in the human system (Acosta-Rodriguez et al., 2007a; Wilson et al., 2007). IL-1β₁, IL-6, and IL-23 production by monocytes and DCs, and the resulting development of human Th17 cells, can be induced by bacterial products, such as LPS or peptidoglycan (Acosta-Rodriguez et al., 2007a; Acosta-Rodriguez et al., 2007b; van Beelen et al., 2007). However, the mechanisms driving Th17 responses in noninfectious settings, such as autoimmunity or cancer, remain unclear.Here, we report that the development of human Th17 cells from naive, effector, and memory CD4⁺ T cell precursors induced by the previously identified Th17-driving cytokines (IL-1β, IL-6, and IL-23) or by IL-1β/IL-6/IL-23-producing MDSCs, is promoted by exogenous NO (or NO produced by human MDSCs) and critically depends on the induction of endogenous NOS2 in differentiating CD4⁺ T cells.     

Combined Evaluation of Commonly Used Techniques,Including PCR,for Diagnosis of Mouse Fur Mites

Our study evaluated and compared the false-negative rates (FNR) of a wide array of fur-mite diagnostic tests, including 2 postmortem tests (pelt exam and sticky paper) and 3 antemortem tests (adhesive tape, fur pluck, and PCR). Past publications examining fur-mite diagnostic techniques primarily used paired comparisons, evaluating tests by their level of agreement with only one other test. However, different combinations or pairs of diagnostics are used in the different studies, making the results of these comparisons difficult to interpret across all available diagnostics. In the current study, mice from a conventionally maintained colony endemic for Myobia musculi were identified as positive based on at least one positive diagnostic test. From this pool of positive animals, the FNR of all tests were quantified. The PCR assay and the pelt exam performed the best, with 0% and 2% FNR respectively, whereas tape, fur-pluck, and sticky-paper tests showed 24%, 26%, and 36% FNR, respectively. Our study shows that for mice in a colony naturally infested with Myobia musculi, PCR testing can be used for reliable antemortem detection, and pelt exam performed by experienced examiners is reliable for postmortem detection.Abbreviation: FNR, false-negative rateFur mites are a common pathogen in laboratory rodents, and infestations usually are caused by Myobia musculi, Myocoptes musculinis, or Radfordia affinis.^1,2,12,13,16 Infestation can result in mite-associated ulcerative dermatitis,^10,34 prolonged inflammatory responses,¹⁸ systemic immune effects,^19-21,26 and decreased reproductive indices.³⁴ Numerous treatment modalities have been reported,^{2-4,9,14,16,25,27,28,31,37} but their success at eradicating outbreaks varies. A survey of institutions conducted in 2008⁵ revealed that 30% and 40% of respondents reported infestations of Myobia musculi and Myocoptes musculinis, respectively, whereas recent prevalence data suggests an overall incidence of 12% in North America.³⁰ Many institutions with barrier facilities exclude fur mites,^1,16,18,34 and policies to reject imports from facilities with endemic or sporadic infestations disrupts collaborations between investigators.¹⁸Low prevalence in barrier housing coupled with poor diagnostic methods makes detection challenging.⁷ Despite the multiple testing methodologies available, successful identification of mite-infested mice remains problematic.^4,32 Soiled-bedding sentinels, the current standard in colony health surveillance, has conflicting reports of reliability in the detection of fur-mite infestations.^23,32 Low cage densities and incorrect sampling site strategies may further decrease detection.²⁴Active infestations in live mice are often diagnosed via transparent adhesive-tape tests,^4,14,25 but they can also be identified by skin scrape, fur pluck, sticky paper, pelt examinations,^2,12 and PCR tests.^3,6,35 A survey of 16 studies^{3,4,9,14,17,18,23-25,29,31-36} involving the diagnosis and treatment of fur mites revealed that the transparent-tape test is the most commonly performed diagnostic test, followed by fur pluck (Figure 1). One group of authors⁴ reported the skin scrape test to be the most reliable method in their experience and gauged other tests by the level of agreement to the skin scrape. Their study⁴ concluded that skin scrape was the most reliable test, followed by pelt examination, transparent adhesive-tape test, fur pluck, and observation. Recent studies suggest direct or environmental sampling PCR can be a sensitive means for detection of mites¹⁷ and that PCR prior to treatment is sensitive for fur mites.³⁵ Other studies^24,35 compared results between 2 selected tests (fur pluck compared with sticky paper;²⁴ transparent adhesive tape compared with PCR³⁵), making it difficult to infer how the evaluated diagnostics compare with other methods not included in the study. When the 5 available comparative fur-mite studies are assessed, it becomes clear that there is little overlap in the diagnostic tests assessed (Figure 2). In the current study, we evaluated 4 commonly used diagnostic methods to enable a side-by-side comparison. Two of those tests were repeated on a second cohort of mice, which also were tested by PCR. This process allowed us to rank the 5 tests used in the study. A false-negative rate (FNR), defined by the occurrence of negative test results in subjects known to be infested, was calculated, instead of agreement between pairs of diagnostics. Here we evaluated the FNR of a wide array of diagnostics, including 2 postmortem and 3 antemortem tests, in the same study.Open in a separate windowFigure 1.Number of publications using various fur-mite diagnostic techniques in studies. Results were compiled by reviewing 16 studies evaluating fur-mite diagnosis or treatment, many of which used multiple diagnostic tests.Open in a separate windowFigure 2.Overview of comparative fur-mite diagnostic studies.