Transcriptomic analysis of lung development in wildtype and CFTR-/- sheep suggests an early inflammatory signature in the CF distal lung.

The precise molecular events initiating human lung disease are often poorly characterized. Investigating prenatal events that may underlie lung disease in later life is challenging in man, but insights from the well-characterized sheep model of lung development are valuable. Here, we determine the transcriptomic signature of lung development in wild-type sheep (WT) and use a sheep model of cystic fibrosis (CF) to characterize disease associated changes in gene expression through the pseudoglandular, canalicular, saccular, and alveolar stages of lung growth and differentiation. Using gene ontology process enrichment analysis of differentially expressed genes at each developmental time point, we define changes in biological processes (BP) in proximal and distal lung from WT or CF animals. We also compare divergent BP in WT and CF animals at each time point. Next, we establish the developmental profile of key genes encoding components of ion transport and innate immunity that are pivotal in CF lung disease and validate transcriptomic data by RT-qPCR. Consistent with the known pro-inflammatory phenotype of the CF lung after birth, we observe upregulation of inflammatory response processes in the CF sheep distal lung during the saccular stage of prenatal development. These data suggest early commencement of therapeutic regimens may be beneficial.

Basal Diet Fed to Recipient Mice Was the Driving Factor for Colitis and Colon Tumorigenesis, despite Fecal Microbiota Transfer from Mice with Severe or Mild Disease.

Consumption of the total Western diet (TWD) in mice has been shown to increase gut inflammation, promote colon tumorigenesis, and alter fecal microbiome composition when compared to mice fed a healthy diet, i.e., AIN93G (AIN). However, it is unclear whether the gut microbiome contributes directly to colitis-associated CRC in this model. The objective of this study was to determine whether dynamic fecal microbiota transfer (FMT) from donor mice fed either the AIN basal diet or the TWD would alter colitis symptoms or colitis-associated CRC in recipient mice, which were fed either the AIN diet or the TWD, using a 2 × 2 factorial experiment design. Time-matched FMT from the donor mice fed the TWD did not significantly enhance symptoms of colitis, colon epithelial inflammation, mucosal injury, or colon tumor burden in the recipient mice fed the AIN diet. Conversely, FMT from the AIN-fed donors did not impart a protective effect on the recipient mice fed the TWD. Likewise, the composition of fecal microbiomes of the recipient mice was also affected to a much greater extent by the diet they consumed than by the source of FMT. In summary, FMT from the donor mice fed either basal diet with differing colitis or tumor outcomes did not shift colitis symptoms or colon tumorigenesis in the recipient mice, regardless of the basal diet they consumed. These observations suggest that the gut microbiome may not contribute directly to the development of disease in this animal model.

Early developmental phenotypes in the cystic fibrosis sheep model.

Highly effective modulator therapies for cystic fibrosis (CF) make it a treatable condition for many people. However, although CF respiratory illness occurs after birth, other organ systems particularly in the digestive tract are damaged before birth. We use an ovine model of CF to investigate the in utero origins of CF disease since the sheep closely mirrors critical aspects of human development. Wildtype (WT) and CFTR -/- sheep tissues were collected at 50, 65, 80, 100, and 120 days of gestation and term (147 days) and used for histological, electrophysiological, and molecular analysis. Histological abnormalities are evident in CFTR-/- -/-  animals by 80 days of gestation, equivalent to 21 weeks in humans. Acinar and ductal dilation, mucus obstruction, and fibrosis are observed in the pancreas; biliary fibrosis, cholestasis, and gallbladder hypoplasia in the liver; and intestinal meconium obstruction, as seen at birth in all large animal models of CF. Concurrently, cystic fibrosis transmembrane conductance regulator (CFTR)-dependent short circuit current is present in WT tracheal epithelium by 80 days gestation and is absent from CFTR -/- tissues. Transcriptomic profiles of tracheal tissues confirm the early expression of CFTR and suggest that its loss does not globally impair tracheal differentiation.

Detection of asinine gammaherpesviruses in association with pulmonary fibrosis in free-ranging donkeys.

A mortality event among recently captured feral donkeys (Equus asinus) occurred in south-central Utah in 2016. The deaths were sporadic, and clinical signs were indicative of respiratory disease, likely associated with an infectious etiology. Ten of 13 donkeys autopsied had moderate-to-severe interstitial fibrosing pneumonia, and one had pyogranulomatous pneumonia. Consensus PCRs directed toward the DNA polymerase and DNA packaging terminase subunit 1 for herpesviruses were performed followed by sequencing of the PCR amplicons and phylogenetic analysis. Asinine herpesvirus 4 (AsHV4) and 5 (AsHV5) were consistently identified in lung tissues of affected donkeys. No other herpesviruses were identified, and herpesviral DNA was not detected in lung tissues of 2 donkeys without evidence of respiratory disease. The detection of asinine gammaherpesviruses may have been associated with the lesions described. AsHV4 and AsHV5 have been reported in previous studies as novel gammaherpesviruses based on sequences obtained from donkeys with interstitial pneumonia and marked syncytial cell formation. Our findings suggest that the association of asinine gammaherpesviruses with respiratory conditions in equids deserves further attention.

The Western Dietary Pattern Combined with Vancomycin-Mediated Changes to the Gut Microbiome Exacerbates Colitis Severity and Colon Tumorigenesis.

Previous work by our group using a mouse model of inflammation-associated colorectal cancer (CAC) showed that the total Western diet (TWD) promoted colon tumor development. Others have also shown that vancomycin-mediated changes to the gut microbiome increased colorectal cancer (CRC). Therefore, the objective of this study was to determine the impact of vancomycin on colon tumorigenesis in the context of a standard mouse diet or the TWD. A 2 × 2 factorial design was used, in which C57Bl/6J mice were fed either the standard AIN93G diet or TWD and with vancomycin in the drinking water or not. While both the TWD and vancomycin treatments independently increased parameters associated with gut inflammation and tumorigenesis compared to AIN93G and plain water controls, mice fed the TWD and treated with vancomycin had significantly increased tumor multiplicity and burden relative to all other treatments. Vancomycin treatment significantly decreased alpha diversity and changed the abundance of several taxa at the phylum, family, and genus levels. Conversely, basal diet had relatively minor effects on the gut microbiome composition. These results support our previous research that the TWD promotes colon tumorigenesis and suggest that vancomycin-induced changes to the gut microbiome are associated with higher tumor rates.

Zika Virus Associated Pathology and Antigen Presence in the Testicle in the Absence of Sexual Transmission During Subacute to Chronic Infection in a Mouse Model.

Zika virus (ZIKV) is an arboviral infection that has been shown to be sexually transmitted. The study outlined herein aims to determine if accessory sex glands and epididymal epithelial cells are sources of viral persistence in subacute and chronic ZIKV infection, and if infection of these organs is important in sexual transmission during long-term (chronic) infection. Male interferon type I receptor knockout (Ifnar-/-) mice were challenged with ZIKV and reproductive tissues were harvested 14 and 35 days post infection (DPI) for inoculation studies and 14, 35 and 70 DPI for histopathology. Artificial insemination fluid derived from epididymal flush and seminal plasma from the prostate and seminal vesicle was obtained from ZIKV inoculated and sham-infected males. Naïve interferon type I and II receptor knockout (AG129) female mice were pre-treated with progesterone and inoculated intravaginally with artificial insemination fluid from ZIKV-infected males. ZIKV RNA was detected in the artificial insemination fluid generated from epididymal flush or seminal plasma from ZIKV infected males at 14 and 35 DPI. ZIKV antigens were only detected in seminiferous tubules at 14 DPI. Epididymal epithelial cells did not show ZIKV antigen immunoreactivity at 14, 35 or 70 DPI. Severe fibrosing orchitis (end stage orchitis) was observed at 35 and 70 DPI. Mild inflammation and peri-tubular fibrosis were observed in the epididymis following clearance of virus. Viral RNA was not detected by PCR in whole blood samples of females from any intravaginal experimental group and only detected in 20% of subcutaneously inoculated animals (derived from 1 experimentally infected male) at 35 DPI. While ZIKV RNA and antigens can be detected in the male reproductive tract at 14 DPI and RNA can also be detected at 35 DPI, intravaginal inoculation of artificial insemination fluid from these time-points failed to result in viremia in naïve females inoculated intravaginally. These studies support the hypothesis that epididymal epithelial cells are critical to sexual transmission in immunocompromised mice. Additionally, acute but not chronic male reproductive tract infection with ZIKV results in infectious virus capable of being sexually transmitted in mice.

Incomplete Urethral Duplication Associated with a Dermoid Cyst within a Vascular Hamartoma in a Female Dog.

A seven-year-old spayed female Labrador retriever presented for necropsy following an acute history of thrombocytopenia, anemia, leukocytosis and abdominal effusion. A 2 × 3 × 10 cm, cylindrical to tubular, mottled red-to-tan mass extended from the caudal pelvic cavity caudally and ventrally under the dermis along the caudal aspect of the left pelvic limb adjacent to the semimembranosus and semitendinosus musculature. Histologic examination of the mass revealed a singular central lumen lined by urothelium that multifocally transitioned into non-keratinizing, stratified squamous epithelium associated with few hair follicles and sweat glands. The lumen was surrounded by a dense collagenous stroma containing numerous, variably sized blood vessels. The combination of lesions was consistent with a diagnosis of incomplete urethral duplication associated with a dermoid cyst and vascular hamartoma. To the authors' knowledge, this is the first report of an incomplete urethral duplication associated with a dermoid cyst within a vascular hamartoma.

A sheep model of cystic fibrosis generated by CRISPR/Cas9 disruption of the CFTR gene.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The major cause of limited life span in CF patients is progressive lung disease. CF models have been generated in 4 species (mice, rats, ferrets, and pigs) to enhance our understanding of the CF pathogenesis. Sheep may be a particularly relevant animal to model CF in humans due to the similarities in lung anatomy and development in the two species. Here, we describe the generation of a sheep model for CF using CRISPR/Cas9 genome editing and somatic cell nuclear transfer (SCNT) techniques. We generated cells with CFTR gene disruption and used them for production of CFTR-/- and CFTR+/- lambs. The newborn CFTR-/- sheep developed severe disease consistent with CF pathology in humans. Of particular relevance were pancreatic fibrosis, intestinal obstruction, and absence of the vas deferens. Also, substantial liver and gallbladder disease may reflect CF liver disease that is evident in humans. The phenotype of CFTR-/- sheep suggests this large animal model will be a useful resource to advance the development of new CF therapeutics. Moreover, the generation of specific human CF disease-associated mutations in sheep may advance personalized medicine for this common genetic disorder.

Intracranial, extraneural ectopic lymph node in a bovine (Bos taurus).

The brain from a field necropsied 8-month-old feedlot heifer presenting with an acute history of depression, lethargy, dyspnoea and anorexia was evaluated grossly and by histopathology. The meninges overlying the left cerebral hemisphere contained a 12 × 26 × 32 mm, dark red, soft, ovoid mass. Histologic examination of this tissue revealed a well-organized lymph node with normal architecture. Organization of reactive lymphoid tissue resembling normal lymph node architecture may occur under chronic stimulation. However, there are no known aggregates of lymphoid tissue present within the cranial vault in any veterinary species. This is the first reported case of an intracranial ectopic lymph node in any species.

Syringomyelia in an Adult American Paint Horse.

Syringomyelia is a form of myelodysplasia defined by the formation of one or more fluid-filled cavities within the spinal cord that do not communicate with the central canal. The defect may be congenital or acquired. Clinical signs correlate to the segment of spinal cord affected and include pain, paresis, proprioceptive deficits, alterations in sensation, scoliosis, and autonomic dysfunction. This report describes the clinical and pathologic changes in a case of acquired syringomyelia in a 10-year-old American Paint Horse mare. The horse had a six-week history of progressive proprioceptive deficits in all four limbs, bilateral pelvic limb ataxia, and muscle fasciculations that were unresponsive to treatment with stall rest, phenylbutazone, and dexamethasone. Syringomyelia was diagnosed postmortem within cervical, thoracic, and lumbar spinal cord segments. Acquired syringomyelia should be considered among differential diagnoses in adult horses displaying progressive neurologic deficits.

Heartland virus infection in hamsters deficient in type I interferon signaling: Protracted disease course ameliorated by favipiravir.

Heartland virus (HRTV) is an emerging tick-borne virus (Bunyaviridae, Phlebovirus) that has caused sporadic cases of human disease in several central and mid-eastern states of America. Animal models of HRTV disease are needed to gain insights into viral pathogenesis and advancing antiviral drug development. Presence of clinical disease following HRTV challenge in hamsters deficient in STAT2 function underscores the important role played by type I interferon-induced antiviral responses. However, the recovery of most of the infected animals suggests that other mechanisms to control infection and limit disease offer substantial protection. The most prominent disease sign with HRTV infection in STAT2 knockout hamsters was dramatic weight loss with clinical laboratory and histopathology demonstrating acute inflammation in the spleen, lymph node, liver and lung. Finally, we show that HRTV disease in hamsters can be prevented by the use of favipiravir, a promising broad-spectrum antiviral in clinical development for the treatment of influenza.

Increased Susceptibility to Atrial Fibrillation Secondary to Atrial Fibrosis in Transgenic Goats Expressing Transforming Growth Factor-ß1.

INTRODUCTION: Large animal models of progressive atrial fibrosis would provide an attractive platform to study relationship between structural and electrical remodeling in atrial fibrillation (AF). Here we established a new transgenic goat model of AF with cardiac specific overexpression of TGF-ß1 and investigated the changes in the cardiac structure and function leading to AF. METHODS AND RESULTS: Transgenic goats with cardiac specific overexpression of constitutively active TGF-ß1 were generated by somatic cell nuclear transfer. We examined myocardial tissue, ECGs, echocardiographic data, and AF susceptibility in transgenic and wild-type control goats. Transgenic goats exhibited significant increase in fibrosis and myocyte diameters in the atria compared to controls, but not in the ventricles. P-wave duration was significantly greater in transgenic animals starting at 12 months of age, but no significant chamber enlargement was detected, suggesting conduction slowing in the atria. Furthermore, this transgenic goat model exhibited a significant increase in AF vulnerability. Six of 8 transgenic goats (75%) were susceptible to AF induction and exhibited sustained AF (>2 minutes), whereas none of 6 controls displayed sustained AF (P < 0.01). Length of induced AF episodes was also significantly greater in the transgenic group compared to controls (687 ± 212.02 seconds vs. 2.50 ± 0.88 seconds, P < 0.0001), but no persistent or permanent AF was observed. CONCLUSION: A novel transgenic goat model with a substrate for AF was generated. In this model, cardiac overexpression of TGF-ß1 led to an increase in fibrosis and myocyte size in the atria, and to progressive P-wave prolongation. We suggest that these factors underlie increased AF susceptibility.

Protection against Chikungunya virus induced arthralgia following prophylactic treatment with adenovirus vectored interferon (mDEF201).

Recent outbreaks of Chikungunya virus (CHIKV) infection have resulted in millions of cases of disease with significant morbidity. No approved antiviral treatments exist for the prevention or treatment of this viral disease. Infection with CHIKV results in a high rate of symptomatic disease that primarily includes a debilitating arthralgia. To model this cardinal disease manifestation, adult DBA/1J mice were challenged with CHIKV by footpad injection. Viremia and hind limb virus titers increased ∼100-fold while spleen virus increased >1000-fold within 1day post-virus infection (dpi). Footpad swelling was measured over a 10-day period, with peak swelling observed between 6 and 7dpi. Histology of the hind leg at the site of virus challenge showed evidence of myositis and synovitis starting on 5dpi. Cytokine profiling of the hind limb at the site of inoculation revealed a biphasic inflammatory response represented by an increase in IL-6, MCP-1, IFN-γ, MIP-1α, RANTES, and IL-17. To investigate the prophylactic capacity of IFN, mice were treated with mDEF201, an adenovirus-vectored IFN-α. Intranasal administration of a single 10(7)pfu/ml dose of mDEF201 administered 21days to 24h prior to infection, significantly reduced footpad swelling, virus titers in the hind leg and spleen, and several inflammatory cytokines. Efficacy was not observed when treatment was initiated 24h after virus challenge. This arthralgia model of CHIKV recapitulates relevant disease features commonly observed in human disease making it applicable to preclinical testing of therapies that target both viral replication and the associated joint disease.

Anchoring hepatic gene expression with development of fibrosis and neoplasia in a toxicant-induced fish model of liver injury.

Fish have been used as laboratory models to study hepatic development and carcinogenesis but not for pathogenesis of hepatic fibrosis. In this study, a dimethylnitrosamine-induced fish model of hepatic injury was developed in Japanese medaka (Oryzias latipes) and gene expression was anchored with the development of hepatic fibrosis and neoplasia. Exposed livers exhibited mild hepatocellular degenerative changes 2 weeks' postexposure. Within 6 weeks, hepatic fibrosis/cirrhosis was evident with development of neoplasia by 10 weeks. Stellate cell activation and development of fibrosis was associated with upregulation of transforming growth factor beta 1 (tgfb1), tgfb receptor 2, mothers against decapentaplegic homolog 3 (smad3a), smad3b, beta-catenin (ctnnb1), myc, matrix metalloproteinase (mmp2), mmp14a, mmp14b, tissue inhibitors of metalloproteinase (timp) 2a, timp2b, timp3, collagen type I alpha 1a (col1a1a), and col1a1b and a less pronounced increase in mmp13 and col4a1 expression. Tgfb receptor I expression was unchanged. Immunohistochemistry suggested that biliary epithelial cells and stellate cells were the main producers of TGF-ß1. This study identified a group of candidate genes likely to be involved in the development of hepatic fibrosis and demonstrated that the TGF-ß pathway likely plays a major role in the pathogenesis. These results support the medaka as a viable fish model of hepatic fibrosis.

Canine cutaneous epitheliotropic T-cell lymphoma with vesiculobullous lesions resembling human bullous mycosis fungoides.

The broad spectrum of clinical signs in canine cutaneous epitheliotropic T-cell lymphoma mimics many inflammatory skin diseases and is a diagnostic challenge. A 13-year-old-male castrated golden retriever crossbred dog presented with multifocal flaccid bullae evolving into deep erosions. A shearing force applied to the skin at the periphery of the erosions caused the epidermis to further slide off the dermis suggesting intraepidermal or subepidermal separation. Systemic signs consisted of profound weight loss and marked respiratory distress. Histologically, the superficial and deep dermis were infiltrated by large, CD3-positive neoplastic lymphocytes and mild epitheliotropism involved the deep epidermis, hair follicle walls and epitrichial sweat glands. There was partial loss of the stratum basale. Bullous lesions consisted of large dermoepidermal and intraepidermal clefts that contained loose accumulations of neutrophils mixed with fewer neoplastic cells in proteinaceous fluid. The lifted epidermis was often devitalized and bordered by hydropic degeneration and partial epidermal collapse. Similar neoplastic lymphocytes formed small masses in the lungs associated with broncho-invasion. Clonal rearrangement analysis of antigen receptor genes in samples from skin and lung lesions using primers specific for canine T-cell receptor gamma (TCRgamma) produced a single-sized amplicon of identical sequence, indicating that both lesions resulted from the expansion of the same neoplastic T-cell population. Macroscopic vesiculobullous lesions with devitalization of the lesional epidermis should be included in the broad spectrum of clinical signs presented by canine cutaneous epitheliotropic T-cell lymphoma.

Mechanical evaluation of various external skeletal fixator-intramedullary pin tie-in configurations using a tubular plastic bone model.

Use of external skeletal fixator-intramedullary pin tie-in (ESF-IM pin tie-in) fixators is an adjustable and effective method of fracture fixation in birds. The objective of this study was to evaluate the elements of the ESF-IM tie-in configuration used in birds. Ten variations of constructs were applied to a plastic bone model with a standard gap. Variants included non-tied and tie-in configurations, use of a 6- or 10-mm acrylic bar or a thermoplastic connecting bar, variation in the placement of the proximal fixation pin, use of 1.1-mm (0.045-in) or 1.6-mm (0.062-in) fixation pins, and configurations of 2, 3, or 4 fixation pins. The various constructs were loaded in bending, torque, and compression, and response variables were determined from resulting load-displacement curves (stiffness, load at 1-mm displacement). Results showed that, by using the tie-in configuration, increasing the diameter of the acrylic connecting bar, and increasing the diameter or number of fixation pins, each significantly increased the stiffness in all assessments. Placing the fixation pin distally in the proximal bone model segment increased the stiffness in bending, and adding a fixation pin to the distal bone model segment increased the stiffness in torque and bending. These results quantified the relative importance of specific parameters that effect stiffness and safe load of the ESF-IM tie-in construct as applied to a plastic bone fracture model.

Mechanical evaluation of external skeletal fixator-intramedullary pin tie-in configurations applied to cadaveral humeri from red-tailed hawks (Buteo jamaicensis).

Use of external skeletal fixator-intramedullary pin (ESF-IM) tie-in fixators is an adjustable and effective method of fracture fixation in birds. The objective of this study was to determine the contribution of each of the following parameters to the compressive and torsional rigidity of an ESF-IM pin tie-in applied to avian bones with an osteotomy gap: (1) varying the fixation pin position in the proximal bone segment and (2) increasing the number of fixation pins in one or both bone segments. ESF-IM pin tie-in constructs were applied to humeri harvested from red-tailed hawks (Buteo jamaicensis) (n=24) that had been euthanatized for clinical reasons. Constructs with a variation in the placement of the proximal fixation pin and with 2, 3, or 4 fixation pins applied to avian bone with an osteotomy gap were loaded to a defined displacement in torque and axial compression. Response variables were determined from resulting load-displacement curves (construct stiffness, load at 1-mm displacement). Increasing the number of fixation pins from 1 to 2 per bone segment significantly increased the stiffness in torque (110%) and compression (60%), and the safe load in torque (107%) and compression (50%). Adding a fixation pin to the distal bone segment to form a 3-pin fixator significantly increased the stiffness (27%) and safe load (20%) in torque but not in axial compression. In the configuration with 2 fixation pins, placing the proximal pin distally in the proximal bone segment significantly increased the stiffness in torque (28%), and the safe load in torque (23%) and in axial compression (32%). Results quantified the relative importance of specific parameters affecting the rigidity of ESF-IM pin tie-in constructs as applied to unstable bone fracture models in birds.