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Ferret systemic coronavirus (FRSCV) causes a highly fatal disease of ferrets (Mustela putorius furo). It is believed to be a mutated variant of ferret enteric coronavirus (FRECV) and has a clinical presentation similar to that of feline infectious peritonitis virus (FIPV) in cats. The interplay of infectious diseases and host genetics will become a greater issue in the research environment as genetically modified species other than rodents become available due to advances in gene editing technology. In this case series, we present the clinical and histopathologic features of a FRSCV outbreak that affected 5 out of 10 ferrets with α-1 antitrypsin knockout (AAT KO) over an approximately 1-y period. Clinical features varied, with the affected ferrets presenting with some combination of wasting, hind limb paralysis, incontinence or sudden death. Multiple ferrets had gross pathologic lesions consistent with FRSCV, but the lesions were typically mild. Microscopic pyogranulomatous inflammation was present in 4 ferrets. Immunohistochemistry using an anti-feline coronavirus antibody that cross reacts with ferret coronavirus confirmed infection of intralesional macrophages in 4 out of 5 animals with suspected FRSCV infection. PCR testing of formalin fixed tissue was negative for all ferrets. PCR testing of feces from healthy wild-type ferrets indicated that the endemic presence of FRECV genotype 2, while PCR surveillance testing of other in-house AAT KO ferrets revealed both enteric coronavirus genotypes 1 and 2. This case series highlights the potential for greater disease incidence in the future as genetically modified ferrets are used more often, and may support exclusion of FRECV and similar viruses from highly susceptible ferret genotypes.

Congestion, granular platelet debris both within macrophage and extracellularly, and neutrophil infiltration in the spleen of a baboon that was euthanized with profound thrombocytopenia.

BACKGROUND: Delta-24-RGD, an oncolytic adenovirus, shows promise against glioblastoma. To enhance virus delivery, we recently demonstrated that human bone marrow-derived mesenchymal stem cells loaded with Delta-24-RGD (hMSC-D24) can eradicate glioblastomas in mouse models. There are no studies examining the safety of endovascular selective intra-arterial (ESIA) infusions of MSC-D24 in large animals simulating human clinical situations. OBJECTIVE: To perform canine preclinical studies testing the feasibility and safety of delivering increasing doses of hMSCs-D24 via ESIA infusions. METHODS: ESIA infusions of hMSC-D24 were performed in the cerebral circulation of 10 normal canines in the target vessels (internal carotid artery [ICA]/P1) via transfemoral approach using commercially available microcatheters. Increasing concentrations of hMSC-D24 or particles (as a positive control) were injected into 1 hemisphere; saline (negative control) was infused contralaterally. Toxicity (particularly embolic stroke) was assessed on postinfusion angiography, diffusion-weighted magnetic resonance imaging, clinical exam, and necropsy. RESULTS: ESIA injections were performed in the ICA (n = 7) or P1 (n = 3). In 2 animals injected with particles (positive control), strokes were detected by all assays. Of 6 canines injected with hMSC-D24 through the anterior circulation, escalating dose from 2 × 10(6) cells/20 mL to 1 × 10(8) cells/10 mL resulted in no strokes. Two animals had ischemic and hemorrhagic strokes after posterior cerebral artery catheterization. A survival experiment of 2 subjects resulted in no complications detected for 24-h before euthanization. CONCLUSION: This novel study simulating ESIA infusion demonstrates that MSCs-D24 can be infused safely at least up to doses of 1 × 10(8) cells/10 mL (10(7) cells/ml) in the canine anterior circulation using commercially available microcatheters. These findings support a clinical trial of ESIA infusion of hMSCs-D24.

Visualization in neuroendovascular interventions relies on biplanar fluoroscopy and digital subtraction. Radiation and contrast-related complications are the two major drawbacks. Angioscopy, direct endoluminal optical visualization, does not require radiation or contrast but has limited utility in neurointerventions due to limitations in size and stiffness. A recently developed microangioscope has the miniaturization and flexibility necessary to navigate small, tortuous intracranial vessels, allowing the practical use of neurovascular angioscopy for the first time. The prototype is a coherent fiber bundle microangioscope embedded in a 0.0165’ microcatheter. In human cadaveric experiments, that the microangioscope is compatible with the human intracranial vasculature beyond the Circle of Willis (eg. navigation into and visualization of the M2 origin). In in vivo swine model experiments, the microangioscope has adequate resolution and illumination to identify and differentiate between various intravascular pathologies (eg. red thrombus versus white thrombus). With flow arrest and irrigation, mechanical thrombectomy (both with stent retriever and direct aspiration), coil embolization, stent deployment, and balloon angioplasty were precisely performed under direct visualization with the microangioscope. After deployment, the microangioscope enables direct inspection of devices, providing complementary information to standard angiography. Imaging quality, illumination, and flexibility of the microangioscope progressively improved through modifications to the image bundle. Based on these results, the microangioscope is compatible with the human distal cerebrovasculature; it also provides adequate direct visualization for neurointerventions. Disclosures V. Srinivasan: None. T. Lazaro: None. P. Cooper: 5; C; Vena Medical, Inc. M. Phillips: 5; C; Vena Medical, Inc. R. Garcia: None. S. Chen: None. J. Johnson: None. J. Burkhardt: None. D. Collins: None. P. Kan: 2; C; Medtronic, Cerenovus.

Pigeons (Columba livia) are used in biomedical research for studies of vision, cognition, neuronal pathways, and spatial orientation. Because there are few commercial laboratory sources, research pigeons are typically acquired from local fancier breeders or bred onsite. For acquired pigeons, the health and vaccine status is often unknown. A juvenile pigeon, born onsite and living in an enclosed outdoor loft, presented with small, bleeding, wart-like lesions on the medial aspects of digits 1 and 4. Topical treatment was initiated. Within a week, 4 fledglings were reported for small, dark papular lesions on the face, head, neck, and beak, and shortly thereafter, 2 additional juvenile pigeons developed similar lesions. The fledglings were euthanized, and histologic examination revealed numerous intralesional eosinophilic cytoplasmic viral inclusions (Bollinger bodies) confirming a diagnosis of poxvirus infection, likely pigeon pox. Although usually self-limiting, pigeon pox can cause moderate to severe lesions in fledgling and juvenile birds. Vaccination with a modified live poxvirus labeled for chickens was used to create herd immunity to pigeon poxvirus. Since vaccination of our entire flock and implementation of more stringent health protocols, all lesions have resolved, and no new lesions have been noted.

Fibrocytes are unique cells with innate and adaptive immune functions, but these mechanisms have not been fully explored. The aim of this study was to explain the mechanism by which adoptive transfer of exogenous fibrocytes improved bacterial clearance and increased sepsis survival. Initial flow cytometry-based, in vitro assays demonstrated phagocytosis by fibrocytes and intracellular bacterial killing was confirmed by direct plating of cell lysates after exposure to live bacteria. Intravenous adoptive transfer of fibrocytes at the time of cecal ligation and puncture (CLP) or two hours after CLP in mice increased survivability. Decreased intraperitoneal bacterial burden was also observed. Quantification of peritoneal cell populations using flow cytometry demonstrated transferred and endogenous fibrocytes were significantly increased after CLP, while macrophage and neutrophil numbers were unchanged. To determine the impact in vivo, fluorescently labeled, killed bacteria were injected i.p. into mice ten hours after CLP or sham surgery ± adoptive transfer. Two hours later, flow cytometry of peritoneal cell populations after CLP alone revealed increased phagocytosis by macrophages, neutrophils and endogenous fibrocytes. Transferred fibrocytes had significantly increased phagocytic activity in the septic peritoneum compared to sham and greater activity than any other cell type. Therefore, adoptive transfer may enhance bacterial clearance in early sepsis through the cumulative effects of endogenous and transferred fibrocytes rather than modulating the function of other endogenous phagocytes. Direct phagocytic activity coupled with previously described influences on T cell responses may explain the benefits of fibrocyte transfer in sepsis.

BackgroundEndovascular technological advances have revolutionized the field of neurovascular surgery and have become the mainstay of treatment for many cerebrovascular pathologies. Digital subtraction angiography (DSA) is the ’gold standard' for visualization of the vasculature and deployment of endovascular devices. Nonetheless, with recent technological advances in optics, angioscopy has emerged as a potentially important adjunct to DSA. Angioscopy can offer direct visualization of the intracranial vasculature, and direct observation and inspection of device deployment. However, previous iterations of this technology have not been sufficiently miniaturized or practical for modern neurointerventional practice.ObjectiveTo describe the evolution, development, and design of a microangioscope that offers both high-quality direct visualization and the miniaturization necessary to navigate in the small intracranial vessels and provide examples of its potential applications in the diagnosis and treatment of cerebrovascular pathologies using an in vivo porcine model.MethodsIn this proof-of-concept study we introduce a novel microangioscope, designed from coherent fiber bundle technology. The microangioscope is smaller than any previously described angioscope, at 1.7 F, while maintaining high-resolution images. A porcine model is used to demonstrate the resolution of the images in vivo.ResultsVideo recordings of the microangioscope show the versatility of the camera mounted on different microcatheters and its ability to navigate external carotid artery branches. The microangioscope is also shown to be able to resolve the subtle differences between red and white thrombi in a porcine model.ConclusionA new microangioscope, based on miniaturized fiber optic technology, offers a potentially revolutionary way to visualize the intracranial vascular space.

Estrogen supplementation is a key component of numerous mouse research models but can adversely affect the urinary system. The goal of this study was to develop a clinical scoring system and identify biomarkers of occult urinary tract lesions prior to the development of systemic illness in mice. Ovariectomized or sham-surgery SCID mice were implanted subcutaneously with a placebo pellet or one containing sustained-release estradiol (0.18 mg 60-d release 17β-estradiol). Mice were assessed twice weekly for 4 to 6 wk by using a clinical scoring system that included body condition, general activity, posture, hair coat, hydration, abdominal distension, urine staining of coat and skin, and ability to urinate. Samples were collected weekly for urinalysis, BUN, creatinine, and serum estradiol levels. Terminal samples were analyzed for histopathologic lesions. Compared with placebo controls, estradiol-supplemented mice had higher serum estradiol levels at weeks 2 and 3; significant differences in total clinical scores by the 3-wk time point; and in body condition, general activity, posture, hair coat, and urine staining scores by the 6-wk terminal time point. Urinary tract lesions included hydronephrosis, pyelonephritis, cystitis, and urolithiasis. All mice with urolithiasis had crystalluria, and 5 of the 6 mice with pyelonephritis or hydroureter had dilute urine (that is, specific gravity less than 1.030). However, these findings were not specific to mice with lesions. A total clinical score of 3.5 (maximum, 24) identified estradiol-supplemented mice with 83% specificity and 50% sensitivity, but no single clinical parameter, biomarker, or the total clinical score accurately predicted occult urinary tract lesions. Considering the lesions we observed, prudence is warranted when using pelleted sustained-release estradiol in mice, and important parameters to monitor for animal health include urine staining, body condition score, urine sediment, and urine specific gravity.

Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism and provided information on the potential participation of adaptor protein complexes in their biogenesis.