Transmucosal administration of pentobarbital and phenytoin solution induces euthanasia in bearded dragons (Pogona vitticeps).

OBJECTIVE: To assess the efficacy of transmucosal euthanasia solution to induce euthanasia. ANIMALS: 6 bearded dragons (Pogona vitticeps). METHODS: An initial dose of euthanasia solution containing pentobarbital and phenytoin sodium was administered transmucosally in conscious lizards (100 mg/kg pentobarbital dose), followed by a second dose 20 minutes later (400 mg/kg pentobarbital dose). The presence of movement, leakage of euthanasia solution, behaviors consistent with oral irritation, respiratory rate, heart rate, palpebral and corneal reflex, and response to noxious stimuli were recorded until death, confirmed by the absence of Doppler cardiac flow and cardiac electrical activity. The time to loss of all parameters was calculated. Postmortem evaluation allowed for histopathologic evaluation of the oral cavity and gastrointestinal tract to detect potential mucosal damage from the alkaline euthanasia solution. RESULTS: The median time to death was 300 minutes (range, 300 to 360 minutes), median time to respiratory arrest was 30 minutes (range, 30 to 50 minutes), and median time to loss of deep pain response was 30 minutes (range, 20 to 50 minutes). Signs consistent with oral irritation occurred in 4 of 6 (66.7%) lizards, including 2 lizards that exhibited whole-body spasms after euthanasia solution administration. Histopathologic changes indicating peracute mucosal ulceration, suspected to be from caustic causes, were identified in 1 (1/6 [16.7%]) lizard. CLINICAL RELEVANCE: Transmucosal euthanasia solution administration resulted in clinical euthanasia within 6 hours. This method should be utilized only after premedication with analgesic and/or anesthetic medications due to the potential for acute mucosal ulceration and behaviors that may be distressing in client-owned animals.

An immune deficient mouse model for mucopolysaccharidosis IIIA (Sanfilippo syndrome).

Mucopolysaccharidosis III (MPSIII, Sanfilippo syndrome) is a devastating lysosomal storage disease that primarily affects the central nervous system. MPSIIIA is caused by loss-of-function mutations in the gene coding for sulfamidase (N-sulfoglucosamine sulfohydrolase/SGSH) resulting in SGSH enzyme deficiency, a buildup of heparin sulfate and subsequent neurodegeneration. There is currently no cure or disease modifying treatment for MPSIIIA. A mouse model for MPSIIIA was characterized in 1999 and later backcrossed onto the C57BL/6 background. In the present study, a novel immune deficient MPSIIIA mouse model (MPSIIIA-TKO) was created by backcrossing the immune competent, C57BL/6 MPSIIIA mouse to an immune deficient mouse model lacking Rag2, CD47 and Il2rg genes. The resulting mouse model has undetectable SGSH activity, exhibits histological changes consistent with MPSIIIA and lacks T cells, B cells and NK cells. This new mouse model has the potential to be extremely useful in testing human cellular therapies in an animal model as it retains the MPSIIIA disease phenotype while tolerating xenotransplantation.

Subcutaneous choriocarcinomas in captive Amargosa voles (Microtus californicus scirpensis).

Spontaneous choriocarcinomas are rare, highly vascular, malignant trophoblastic tumors that occur in humans and animals. This report describes the unusual spontaneous presentation of 4 choriocarcinomas within the subcutaneous tissues of 4, multiparous but nongravid, Amargosa voles (Microtus californicus scirpensis) from a captive breeding colony. Two subcutaneous neoplasms were composed of multifocal discohesive and infiltrative aggregates of medium to large trophoblasts and cytotrophoblasts within a fibrovascular stroma. Neoplastic cells were associated with variably sized thrombi and cavitary areas of hemorrhage and necrosis. Two subcutaneous tumors were predominantly composed of expansile, blood-filled, cystic spaces lined by neoplastic cytotrophoblasts and occasionally contained medium to large trophoblasts. Trophoblasts and cytotrophoblasts were positive for pancytokeratin and cytokeratin 8/18, negative for alpha-fetoprotein, and contained intracytoplasmic Periodic acid-Schiff (PAS)-positive glycogen in all 4 tumors. In species with hemochorial placentation, migration of trophoblasts into maternal circulation with embolization to distant nonreproductive tissues occurs and may explain the unusual subcutaneous distribution of these 4 tumors. The 2 multiloculated paucicellular tumors may represent an early stage of neoplastic transformation. To the authors' knowledge, this is the first report characterizing choriocarcinomas in extrareproductive sites in rodents.

Analysis of genome-wide knockout mouse database identifies candidate ciliopathy genes.

We searched a database of single-gene knockout (KO) mice produced by the International Mouse Phenotyping Consortium (IMPC) to identify candidate ciliopathy genes. We first screened for phenotypes in mouse lines with both ocular and renal or reproductive trait abnormalities. The STRING protein interaction tool was used to identify interactions between known cilia gene products and those encoded by the genes in individual knockout mouse strains in order to generate a list of "candidate ciliopathy genes." From this list, 32 genes encoded proteins predicted to interact with known ciliopathy proteins. Of these, 25 had no previously described roles in ciliary pathobiology. Histological and morphological evidence of phenotypes found in ciliopathies in knockout mouse lines are presented as examples (genes Abi2, Wdr62, Ap4e1, Dync1li1, and Prkab1). Phenotyping data and descriptions generated on IMPC mouse line are useful for mechanistic studies, target discovery, rare disease diagnosis, and preclinical therapeutic development trials. Here we demonstrate the effective use of the IMPC phenotype data to uncover genes with no previous role in ciliary biology, which may be clinically relevant for identification of novel disease genes implicated in ciliopathies.

Isolation and characterization of Lactococcus garvieae from rainbow trout, Onchorhyncus mykiss, from California, USA.

Lactococcus garvieae is an emergent bacterial pathogen of salmonid fish in North America that causes acute infections particularly at water temperatures above 15°C. During 2020, L. garvieae was detected in rainbow trout, Onchorhyncus mykiss, cultured in Southern California and the Eastern Sierras. Infected fish exhibited high mortalities and nonspecific clinical signs of lethargy, erratic swimming, dark skin pigmentation, and exophthalmia. Macroscopic changes included external and internal hemorrhages, mainly in the eyes, liver, coelomic fat, intestine, and brain. Histological examination revealed splenitis, branchitis, panophthalmitis, hepatitis, enteritis, and coelomitis, with variable degrees of tissue damage among evaluated fish. Pure colonies of L. garvieae were isolated from infected trout and specific PCR primers for L. garvieae confirmed the preliminary diagnosis. Multilocus sequence analysis showed that the strains recovered from diseased trout represent a novel genetic group. Isolates were able to form biofilms within 24 h that increased their resistance to disinfection by hydrogen peroxide. Laboratory challenge methods for inducing lactococcosis in steelhead trout, O. mykiss, were evaluated by intracoelomic injection with serial dilutions of L. garvieae. The median lethal dose 21 days post challenge was ∼20 colony-forming units/fish. Experimentally infected trout presented similar clinical signs, gross changes, and microscopic lesions as those with natural disease, fulfilling Koch's postulates and demonstrating the high virulence of the recovered strains.

Retinal degeneration in mice and humans with neuronal ceroid lipofuscinosis type 8.

BACKGROUND: Ceroid lipofuscinosis type 8 belongs to a heterogenous group of vision and life-threatening neurodegenerative diseases, neuronal ceroid lipofuscinosis (NCL). Effective therapy is limited to a single drug for treatment of ceroid lipofuscinosis type 2, necessitating animal disease models to facilitate further therapeutic development. Murine models are advantageous for therapeutic development due to easy genetic manipulation and rapid breeding, however appropriate genetic models need to be identified and characterized before being used for therapy testing. To date, murine models of ocular disease associated with ceroid lipofuscinosis type 8 have only been characterized in motor neuron degeneration mice. METHODS: Cln8-/- mice were produced by CRISPR/Cas9 genome editing through the International Mouse Phenotyping Consortium. Ophthalmic examination, optical coherence tomography, electroretinography, and ocular histology was performed on Cln8-/- mice and controls at 16 weeks of age. Quantification of all retinal layers, retinal pigmented epithelium, and the choriocapillaris was performed using images acquired with ocular coherence tomography and planimetry of histologic sections. Necropsy was performed to investigate concurrent systemic abnormalities. Clinical correlation with human patients with CLN8-associated retinopathy is provided. RESULTS: Retinal degeneration characterized by retinal pigment epithelium mottling, scattered drusen, and retinal vascular attenuation was noted in all Cln8-/- mice. Loss of inner and outer photoreceptor segment demarcation was noted on optical coherence tomography, with significant thinning of the whole retina (P=1e-9), outer nuclear layer (P=1e-9), and combined photoreceptor segments (P=1e-9). A global reduction in scotopic and photopic electroretinographic waveforms was noted in all Cln8-/- mice. Slight thickening of the inner plexiform layer (P=0.02) and inner nuclear layer (P=0.004), with significant thinning of the whole retina (P=0.03), outer nuclear layer (P=0.01), and outer photoreceptor segments (P=0.001) was appreciated on histologic sections. Scattered lipid vacuoles were noted in splenic red pulp of all Cln8-/- mice, though no gross systemic abnormalities were detected on necropsy. Retinal findings are consistent with those seen in patients with ceroid lipofuscinosis type 8. CONCLUSIONS: This study provides detailed clinical characterization of retinopathy in adult Cln8-/- mice. Findings suggest that Cln8-/- mice may provide a useful murine model for development of novel therapeutics needed for treating ocular disease in patients with ceroid lipofuscinosis type 8.

The geropathology of organ-specific aging.

Aging is a complex multidimensional process of progressive decline affecting multiple organ systems by a number of processes that are still not well understood. While many studies have focused on the approach of studying aging across multiple organs, assessment of the contribution of individual organs to overall aging processes is under appreciated. The ability to study and compare organs in the context of organismal aging has been documented recently using a geropathology grading platform in laboratory mice. This concept consists of identifying and grading age-related histologic lesions within organs to generate a quantitative lesion score for each organ, which is representative of the presence and degree of organ-related pathology, and can be compared to scores from other organs examined. This geropathology approach provides a powerful tool to elucidate the basic mechanisms of aging in multiple organs, as well as the response of organs to therapeutic interventions. Furthermore, ongoing work with the concept has expanded and adapted the geropathology grading system to other preclinical animal model species that are commonly used to understand disease associated phenotypes in aging humans, ultimately adding to the utility of the concept.

Spontaneous Disease and Pathology of Naked Mole-Rats.

Naked mole-rats are highly valuable research models and popular exhibition animals at zoos worldwide. Here, we provide comprehensive descriptions of common postmortem findings of naked mole-rats from both research colonies and populations managed in zoological institutions. Included are brief reviews of their natural history and related physiologic adaptations, unique anatomical features, gross and histologic lesions of common as well as rarely reported disease processes, and discussions of possible pathogeneses with recommendations for future investigations to fill knowledge gaps. Based on postmortem data of several hundreds of naked mole-rats in managed care, it is clear that cancer is extremely rare and infectious disease is infrequently reported. However, despite relatively benign aging phenotypes in this species, several degenerative processes have been nevertheless observed in older populations of naked mole-rats. As such, some potential diet and husbandry-related issues are discussed in addition to the one of the most prominent causes of morbidity and mortality, conspecific aggression and traumas. From this review of lesions and disease, it is clear that pathology, including histopathology, is integral to better understanding mechanisms of healthy aging and cancer resistance of these extraordinary rodents.

Injection Reactions after Administration of Sustained-release Meloxicam to BALB/cJ, C57BL/6J, and Crl:CD1(ICR) Mice.

The sustained-release formulation of meloxicam (MSR) is a compounded NSAID that may provide pain relief for as long as 72 h after administration. MSR injection-site skin reactions have occurred in several species but have not previously been observed in mice. We investigated the development and progression of localized skin reactions after a single injection of MSR in Crl:CD1(ICR), C57BL/6J, and BALB/cJ mice. Each mouse received a subcutaneous injection of MSR (n = 60), standard-formulation meloxicam (MEL; n = 24) or saline (control; SC; n = 24) and was scored daily according to a 5-point system for erythema and mass characteristics. Mice were euthanized at either 7 or 14 d after injection and underwent postmortem analysis. MSR-treated mice had more erythematous and mass reactions than did MEL and SC mice. Mass lesions developed in 49 MSR mice (82%; 95% CI, 70% to 90%), 5 MEL animals (21%; 95% CI, 7% to 42%), and 1 SC mouse. MSR-treated BALB/cJ developed erythematous lesions less frequently than similarly treated Crl:CD1(ICR) or C57BL/6J. Lesions often were ventrolateral to the injection site. The median times to the appearance of mass and erythematous lesions were 2 d and 3 d, respectively. Histologically, the erythematous and mass reactions correlated with necrotizing to pyogranulomatous injection-site panniculitis. Inflammation severity scores at 7 and 14 d after injection were greater in the MSR-treated group than the other 2 groups. No strain- or sex-associated differences emerged except that inflammation severity scores at day 14 were higher in Crl:CD1(ICR) females than males. The character of the inflammatory response in MSR-treated mice did not differ between 7 and 14 d after injection, indicating that MSR-induced inflammation is slow to resolve. The ventral migration and delayed onset of MSR injection-site reactions could result in their being attributed to another cause or not being identified. Researchers and clinicians should be aware of the potential for slowly resolving injection-site reactions with MSR.

Development of Mast Cell and Eosinophil Hyperplasia and HLH/MAS-Like Disease in NSG-SGM3 Mice Receiving Human CD34+ Hematopoietic Stem Cells or Patient-Derived Leukemia Xenografts.

Immunocompromised mouse strains expressing human transgenes are being increasingly used in biomedical research. The genetic modifications in these mice cause various cellular responses, resulting in histologic features unique to each strain. The NSG-SGM3 mouse strain is similar to the commonly used NSG (NOD scid gamma) strain but expresses human transgenes encoding stem cell factor (also known as KIT ligand), granulocyte-macrophage colony-stimulating factor, and interleukin 3. This report describes 3 histopathologic features seen in these mice when they are unmanipulated or after transplantation with human CD34+ hematopoietic stem cells (HSCs), virally transduced hCD34+ HSCs, or a leukemia patient-derived xenograft. The first feature is mast cell hyperplasia: unmanipulated, naïve mice develop periductular pancreatic aggregates of murine mast cells, whereas mice given the aforementioned human cells develop a proliferative infiltrative interstitial pancreatic mast cell hyperplasia but with human mast cells. The second feature is the predisposition of NSG-SGM3 mice given these human cells to develop eosinophil hyperplasia. The third feature, secondary hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS)-like disease, is the most pronounced in both its clinical and histopathologic presentations. As part of this disease, a small number of mice also have histiocytic infiltration of the brain and spinal cord with subsequent neurologic or vestibular signs. The presence of any of these features can confound accurate histopathologic interpretation; therefore, it is important to recognize them as strain characteristics and to differentiate them from what may be experimentally induced in the model being studied.

Development of a Geropathology Grading Platform for nonhuman primates.

A geropathology grading platform (GGP) for assessing age-related lesions has been established and validated for in inbred strain of mice. Because nonhuman primates (NHPs) share significant similarities in aging and spontaneous chronic diseases with humans, they provide excellent translational value for correlating histopathology with biological and pathological events associated with increasing age. Descriptive age-associated pathology has been described for rhesus macaques and marmosets, but a grading platform similar to the mouse GGP does not exist. The value of these NHP models is enhanced by considerable historical data from clinical, bio-behavioral, and social domains that align with health span in these animals. Successful adaptation of the mouse GGP for NHPs will include 1) expanding the range of organs examined; 2) standardizing necropsy collection, tissue trimming, and descriptive lesion terminology; 3) expanding beyond rhesus macaques and marmosets to include other commonly used NHPs in research; and 4) creating a national resource for age-related pathology to complement the extensive in-life datasets. Adaptation of the GGP to include translational models other than mice will be crucial to advance geropathology designed to enhance aging research.

Pharmacokinetic and Efficacy Study of Acyclovir Against Cyprinid Herpesvirus 3 in Cyprinus carpio.

Cyprinid Herpesvirus 3 (CyHV-3), more commonly known as Koi Herpesvirus (KHV), is a re-emergent virus causing acute systemic infection with high mortality rates in koi fish (Cyprinus carpio). Survivors from outbreaks can become latent carriers, with viral reactivation under stressful conditions and permissible temperatures. No vaccines or treatments are currently available in the United States. Acyclovir has been shown effective in vitro against KHV. This study aimed to evaluate the cytotoxicity of acyclovir and cidofovir to koi fin (KF1) cells, the efficacy of a single antiviral intracoelomic dose in a koi fingerling cohabitation challenge, and the pharmacokinetics of the effective antiviral. Initially, a lactate dehydrogenase release-based assay revealed no significant acyclovir or cidofovir cytotoxicity to KF1 cells for 24 h at up to 1,500 µM. In laboratory-controlled challenges, KHV associated mortalities occurred 2 weeks post-infection. At this point, fish were treated with an antiviral (10 mg/kg acyclovir or 5 mg/kg cidofovir) or sterile phosphate-buffered solution. Morbidity and mortality were monitored for 30 days. A significant cumulative mortality reduction (p ≤ 0.05), and a 3-day mortality delay were detected in the acyclovir-treated group. Similar viral loads were detected in gills recovered from mortalities throughout the challenge and surviving fish at the end of the challenge regardless of treatment. For pharmacokinetic analysis, blood was collected at various timepoints after acyclovir administration. Liquid chromatography tandem mass spectrometry plasma analysis indicated a 141 µM peak plasma concentration at 0.75 h, a 14 h half-life, and a 0.05/h elimination rate constant. Histopathology of target tissues detected no evidence of acyclovir toxicity. Results suggest that a single 10 mg/kg dose of acyclovir administered intracoelomically to koi fingerlings is safe and reduces cumulative mortality during a KHV mortality event. However, multiple doses are probably required for effective treatment of pet fish.

Injection-site Reactions to Sustained-release Meloxicam in Sprague-Dawley Rats.

An extended-release formulation of the NSAID meloxicam (MSR) is used to provide 72 h of continuous analgesia in many species, including rodents. Although standard formulations of meloxicam are frequently used in rats with no observable injection-site reactions, the potential adverse effects from MSR have not been characterized sufficiently nor has a prospective study of these effects been performed in rats. To address this deficiency, we evaluated injection-site reactions after a single subcutaneous administration of MSR (n = 16) or sterile saline (SC, n = 6) in the flank of age- and sex-matched Sprague-Dawley rats. Mass and erythema scores were measured daily for 2 wk, and injection sites were collected for histopathology after euthanasia. Rats were randomly selected for euthanasia at 7 d (n = 12) or 14 d (n = 10) after injection to capture the subacute and chronic phases of mass and erythematic lesion formation. No rats in the SC group developed lesions, whereas all 16 MSR-treated rats developed masses. The median time to first mass in the MSR treatment group was 3 d (95% CI, 2-3 d), and nearly 8 d for erythema (95% CI, 6.7-9.1 d). The trajectory of mass lesion severity showed rapid progression from score 1 at onset (day 2 or 3) to score 2 for almost all animals by day 5 or 6. Histopathology was characterized by localized inflammation with central necrosis and peripheral fibrosis, with some sections showing developing draining tracts. Given the high prevalence and severity of localized skin reactions, MSR analgesia should be considered carefully for Sprague-Dawley rats.

Pathology of Diseases of Geriatric Exotic Mammals.

The review covers select disease conditions most frequently described in aging rodents (rats, mice, hamsters, guinea pigs), rabbits, and ferrets. The conditions are categorized by general organ systems, infectious diseases, and neoplasms. Two data systems, the Veterinary Medical Teaching Hospital and Comparative Pathology Laboratory at the University of California, Davis and Zoo/Exotic Pathology Service, Citrus Heights, California were used in the determining disease conditions to describe.

DISEASE AND PATHOLOGICAL CONDITIONS OF AN ENDANGERED RODENT, MICROTUS CALIFORNICUS SCIRPENSIS, IN A CAPTIVE-REARING FACILITY AND IN THE WILD.

Causes of morbidity and mortality and a survey of infectious disease agents were collated from wild and colony-raised endangered Amargosa voles (Microtus californicus scirpensis). Six voles from the wild and 295 voles in the captive-breeding colony were included in the study upon identification of an infectious agent during screening, identification of clinical signs of disease, or finding a pathological condition or infectious agent on necropsy. Findings included 28 significant or incidental pathological conditions of seven organ systems and 19 parasitic, viral, bacterial, or fungal agents. Several voles captured in the wild had fungal osteomyelitis of the tail that disseminated systemically in a vole brought from the wild to the colony and may have been caused by a Penicillium sp. Three voles reintroduced from the colony to the wild experienced inanition and subsequent severe hepatic and moderate renal tubular lipidosis. The most common significant pathological conditions in colony-reared voles were chronic interstitial nephritis with proteinosis; cardiomyopathy; trichobezoars that, in intestines or cecocolic junctions, sometimes induced local rupture or infarction with peritonitis; multifocal gastrointestinal ulceration and colibacillosis; acute renal tubular necrosis or nephritis; sepsis; hepatic and renal lipidosis; molar apical elongation sometimes progressing to invasion of the calvarium; and mammary tumors. Uncommon diagnoses included intervertebral disc disease; microvascular dysplasia; and multifocal bacterial abscessation. Common or clinically important infectious agents included Demodex sp. mites in hair follicles, Demodex sp. in esophageal mucosa, and an outbreak of tropical rat mites thought to have been introduced via the straw bedding; gastrointestinal Helicobacter sp.; attaching and effacing Escherichia coli; and Citrobacter braakii, a possible zoonotic bacterium. This survey of species-specific diseases and pathogens was possible because the established health surveillance program that is part of the species recovery plan allowed for monitoring of voles throughout the duration of their natural life spans in captivity.

Single-cell transcriptomics of the naked mole-rat reveals unexpected features of mammalian immunity.

The immune system comprises a complex network of specialized cells that protects against infection, eliminates cancerous cells, and regulates tissue repair, thus serving a critical role in homeostasis, health span, and life span. The subterranean-dwelling naked mole-rat (NM-R; Heterocephalus glaber) exhibits prolonged life span relative to its body size, is unusually cancer resistant, and manifests few physiological or molecular changes with advancing age. We therefore hypothesized that the immune system of NM-Rs evolved unique features that confer enhanced cancer immunosurveillance and prevent the age-associated decline in homeostasis. Using single-cell RNA-sequencing (scRNA-seq) we mapped the immune system of the NM-R and compared it to that of the short-lived, cancer-prone mouse. In contrast to the mouse, we find that the NM-R immune system is characterized by a high myeloid-to-lymphoid cell ratio that includes a novel, lipopolysaccharide (LPS)-responsive, granulocyte cell subset. Surprisingly, we also find that NM-Rs lack canonical natural killer (NK) cells. Our comparative genomics analyses support this finding, showing that the NM-R genome lacks an expanded gene family that controls NK cell function in several other species. Furthermore, we reconstructed the evolutionary history that likely led to this genomic state. The NM-R thus challenges our current understanding of mammalian immunity, favoring an atypical, myeloid-biased mode of innate immunosurveillance, which may contribute to its remarkable health span.

Experimental Infection of Mice with Veronaea botryosa as a Model for Human Phaeohyphomycosis.

Veronaea botryosa is a ubiquitous, dematiaceous mold capable of causing cutaneous and subcutaneous lesions in humans. In the last decade, V. botryosa has been associated with emergent systemic fungal infections in aquatic animals, including cultured sturgeon (Acipenser spp.), captive amphibians, and wild reptiles. Recently, repetitive extragenic palindromic PCR (rep-PCR) fingerprinting has demonstrated intraspecific variability among V. botryosa isolates from different clinically affected hosts and geographic regions. However, little is known regarding the pathogenic potential of the different genetic clades, and no mammalian model currently exists to investigate V. botryosa phaeohyphomycosis. In this study, we inoculated immunocompetent heterozygotic (nu/+) and immunodeficient homozygotic (nu/nu) Hsd:Athymic Nude-Fox1nu mice subcutaneously or through orogastric gavage with 1 of 3 representative V. botryosa strains that had been recovered from white sturgeon (Acipenser transmontanus), green sea turtle (Chelonia mydas), and human hosts and typed by using rep-PCR analysis. Daily mortality and morbidity were recorded, and dissemination of the fungus was investigated through culture of splenic samples and histologic analysis of the injection site, regional lymph nodes, salivary gland, spleen, liver, mesenteric lymph node, and gastrointestinal tract. No differences in survival, fungal burden, or dissemination were observed between fungal strains, routes of inoculation, or host immune status. Fungal infection was observed after subcutaneous inoculation only, was localized to the inoculation site, and was identified in both nu/nu and nu/+ mice. Fungal strain variability was not associated with virulence in a murine model of infection, and this novel mouse model of V. botryosa phaeohyphomycosis recapitulates the human clinical condition.

Validation of a geropathology grading system for aging mouse studies.

An understanding of early-onset mechanisms underlying age-related changes can be obtained by evaluating changes that precede frailty and end of life using histological characterization of age-related lesions. Histopathology-based information as a component of aging studies in mice can complement and add context to molecular, cellular, and physiologic data, but there is a lack of information regarding scoring criteria and lesion grading guidelines. This report describes the validation of a grading system, designated as the geropathology grading platform (GGP), which generated a composite lesion score (CLS) for comparison of histological lesion scores in tissues from aging mice. To assess reproducibility of the scoring system, multiple veterinary pathologists independently scored the same slides from the heart, lung, liver, and kidney from two different strains (C57BL/6 and CB6F1) of male mice at 8, 16, 24, and 32 months of age. There was moderate to high agreement between pathologists, particularly when agreement within a 1-point range was considered. CLS for all organs was significantly higher in older versus younger mice, suggesting that the GGP was reliable for detecting age-related pathology in mice. The overall results suggest that the GGP guidelines reliably distinguish between younger and older mice and may therefore be accurate in distinguishing between experimental groups of mice with more, or less, age-related pathology.

Generality of Post-Antimicrobial Treatment Persistence of Borrelia burgdorferi Strains N40 and B31 in Genetically Susceptible and Resistant Mouse Strains.

A basic feature of infection caused by Borrelia burgdorferi, the etiological agent of Lyme borreliosis, is that persistent infection is the rule in its many hosts. The ability to persist and evade host immune clearance poses a challenge to effective antimicrobial treatment. A link between therapy failure and the presence of persister cells has started to emerge. There is growing experimental evidence that viable but noncultivable spirochetes persist following treatment with several different antimicrobial agents. The current study utilized the mouse model to evaluate if persistence occurs following antimicrobial treatment in disease-susceptible (C3H/HeJ [C3H]) and disease-resistant (C57BL/6 [B6]) mouse strains infected with B. burgdorferi strains N40 and B31 and to confirm the generality of this phenomenon, as well as to assess the persisters' clinical relevance. The status of infection was evaluated at 12 and 18 months after treatment. The results demonstrated that persistent spirochetes remain viable for up to 18 months following treatment, as well as being noncultivable. The phenomenon of persistence in disease-susceptible C3H mice is equally evident in disease-resistant B6 mice and not unique to any particular B. burgdorferi strain. The results also demonstrate that, following antimicrobial treatment, both strains of B. burgdorferi, N40 and B31, lose one or more plasmids. The study demonstrated that noncultivable spirochetes can persist in a host following antimicrobial treatment for a long time but did not demonstrate their clinical relevance in a mouse model of chronic infection. The clinical relevance of persistent spirochetes beyond 18 months following antimicrobial treatment requires further studies in other animal models.

Noncoding deletions reveal a gene that is critical for intestinal function.

Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants1,2. Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.