Reduced retinal transduction and enhanced transgene-directed immunogenicity with intravitreal delivery of rAAV following posterior vitrectomy in dogs.

Adeno-associated virus (AAV) vector-based gene therapy is a promising treatment strategy for delivery of neurotrophic transgenes to retinal ganglion cells (RGCs) in glaucoma patients. Retinal distribution of transgene expression following intravitreal injection (IVT) of AAV is variable in animal models and the vitreous humor may represent a barrier to initial vector penetration. The primary goal of our study was to investigate the effect of prior core vitrectomy with posterior hyaloid membrane peeling on pattern and efficiency of transduction of a capsid amino acid substituted AAV2 vector, carrying the green fluorescent protein (GFP) reporter transgene following IVT in dogs. When progressive intraocular inflammation developed starting 4 weeks post IVT, the study plan was modified to allow detailed characterization of the etiology as a secondary goal. Unexpectedly, surgical vitrectomy was found to significantly limit transduction, whereas in non-vitrectomized eyes transduction efficiency reached upwards to 37.3% of RGC layer cells. The developing retinitis was characterized by mononuclear cell infiltrates resulting from a delayed-type hypersensitivity reaction, which we suspect was directed at the GFP transgene. Our results, in a canine large animal model, support caution when considering surgical vitrectomy before IVT for retinal gene therapy in patients, as prior vitrectomy appears to significantly reduce transduction efficiency and may predispose the patient to development of vector-induced immune reactions.

Immunohistochemical characterization and morphometric analysis of macrophages in rat mammary tumors.

Macrophages are an important leukocyte component of the microenvironment of neoplasms. Macrophages have classically been subdivided into M1 and M2, depending on their roles in immune response, wound healing, and promotion or inhibition of tumor growth. In human breast cancer, increased presence of M2 macrophages has been associated with poor prognosis. The authors hypothesized that rat mammary carcinomas have increased macrophage influx compared to benign mammary proliferative lesions and normal mammary glands as well. In humans, both M1 and M2 macrophages express CD68, while CD163 is expressed primarily by M2 macrophages. Based on a single immunolabeling protocol with anti-CD68 and anti-CD163 antibodies, the extent of macrophage influx was investigated by morphometry to quantitate the immunopositive cells in normal rat mammary glands, benign mammary proliferative lesions, and mammary carcinomas. In mammary carcinomas, there was significantly higher percentage of CD68+ cells compared to benign mammary proliferative lesions and normal mammary glands. There was also higher percentage of CD163+ cells in mammary carcinomas compared to benign mammary proliferative lesions. Thus, increase in CD68+ and CD163+ macrophages corresponded to increased malignancy of rat mammary tumors in this study.

Megaesophagus in Friesian horses associated with muscular hypertrophy of the caudal esophagus.

Friesian horses have a perceived high rate of congenital or hereditary diseases, including megaesophagus, that may lead to choke and death. A retrospective study was performed to determine the prevalence and pathologic characteristics of esophageal disease in 852 horses, including 17 Friesians, that had been necropsied over a 6-year period at the Diagnostic Center for Population and Animal Health. Forty-two horses had grossly described esophageal lesions (25 muscular hypertrophy, 7 hemorrhage, 6 megaesophagus, 4 erosion/ulceration, 3 obstruction, 2 tears, 2 secondary neoplasms, 2 lymphoid patches, 1 thin wall, 1 esophagitis). Some of these lesions occurred concurrently in the same horse. Ten of these horses died or were euthanatized because of severe esophageal disease (6 megaesophagus causing tears in 2 horses, 3 esophageal obstruction with food bolus, and 1 esophagitis). All 6 horses with megaesophagus were Friesians. No cause for megaesophagus was noted in the necropsy reports; however, 5 of these 6 Friesians had marked caudal esophageal muscular hypertrophy (wall thickness: 1.9 ± 0.3 cm). Microscopic review of the esophagus of these Friesians confirmed smooth muscle hypertrophy, with no obvious fibrosis, degeneration, or loss of myenteric plexi. Unlike the Friesians, the 4 non-Friesian horses with severe esophageal disease had esophageal obstruction with an intraluminal food bolus or severe esophagitis. None had caudal esophageal muscular hypertrophy. It is concluded that in comparison to other horse breeds, Friesians have a higher prevalence of severe esophageal disease, specifically megaesophagus, that is commonly associated with marked caudal muscular hypertrophy.

Beyond H&E: integration of nucleic acid-based analyses into diagnostic pathology.

Veterinary pathology of infectious, particularly viral, and neoplastic diseases has advanced significantly with the advent of newer molecular methodologies that can detect nucleic acid of infectious agents within microscopic lesions, differentiate neoplastic from nonneoplastic cells, or determine the suitability of a targeted therapy by detecting specific mutations in certain cancers. Polymerase chain reaction-based amplification of DNA or RNA and in situ hybridization are currently the most commonly used methods for nucleic acid detection. In contrast, the main methodology used for protein detection within microscopic lesions is immunohistochemistry. Other methods that allow for analysis of nucleic acids within a particular cell type or individual cells, such as laser capture microdissection, are also available in some laboratories. This review gives an overview of the factors that influence the accurate analysis of nucleic acids in formalin-fixed tissues, as well as of different approaches to detect such targets.

Cutaneous mast cell tumor with epitheliotropism in 3 dogs.

Epitheliotropism is an important diagnostic feature of cutaneous epitheliotropic lymphoma and canine cutaneous histiocytoma; however, although noted in certain feline mastocytic diseases, it has not been considered a feature of canine cutaneous mast cell tumor. In this study, 3 canine cutaneous mast cell tumors had epitheliotropic invasion of neoplastic mast cells into the epidermis and follicular epithelium. This unusual histologic finding was characterized by infiltrates of individual and clusters of neoplastic mast cells in the stratum basale and stratum spinosum. The mast cell origin of these cells was documented by demonstration of metachromasia with Giemsa stain and positive immunoreactivity to KIT protein. On the basis of these findings, mast cell tumors should be included in the differential diagnosis for canine cutaneous round cell neoplasms that infiltrate the epidermis.

Somatotroph pituitary tumors in budgerigars (Melopsittacus undulatus).

A series of 11 pituitary tumors in budgerigars were classified on the basis of their clinical, gross, microscopic, and immunohistochemical characteristics. Affected birds were young to middle-aged. Clinically, neurologic signs--including difficulties flying, ataxia, and blindness--were most commonly reported. Additional clinical signs included weight loss, abnormal feathers or molting, increased respiratory efforts, and exophthalmos. Nine birds were diagnosed with chromophobic pituitary adenomas, and 2 birds had chromophobic pituitary carcinomas. Only 1 tumor was delimited to the pituitary gland; the other 10 variably invaded the brain, skull, and retrobulbar space. Distant metastases were identified in 2 birds. All tumors were immunohistochemically strongly positive for growth hormone, consistent with the diagnosis of somatotroph tumors. The common occurrence and early onset may suggest a genetic predisposition of budgerigars to develop somatotroph pituitary tumors with a high incidence of local invasion and with metastatic potential.

Vascular lesions in pigs experimentally infected with porcine circovirus type 2 serogroup B.

Vasculitis is a hallmark lesion of the severe form of systemic porcine circovirus-associated disease (PCVAD). In 2 experimental studies with porcine circovirus type 2 serogroup b (PCV2b), 2 pigs developed fatal PCVAD with acute vasculitis, and 5 related pigs developed chronic lymphohistiocytic and plasmacytic peri- and endarteritis. Five of these pigs (1 with acute vasculitis and 4 with chronic vasculitis) had also been inoculated with bovine viral diarrhea virus type 1 (BVDV1) or BVDV1-like virus. Vascular lesions were similar, independent of whether pigs had been inoculated singly with PCV2b or dually with PCV2b and BVDV1 or BVDV1-like virus. The acute vasculitis was accompanied by marked pulmonary and mesenteric edema and pleural effusion. In situ hybridization demonstrated abundant intracytoplasmic porcine circovirus type 2 (PCV2) nucleic acid in endothelial, smooth muscle-like, and inflammatory cells within and around affected arteries. The pigs with lymphohistiocytic and plasmacytic vasculitis had lesions of systemic PCVAD, including multisystemic lymphoplasmacytic and histiocytic or granulomatous inflammation. PCV2 nucleic acid was detected in renal tubule epithelial cells, mononuclear inflammatory cells, and rare endothelial cells in noninflamed vessels in multiple tissues of these animals. The 2 pigs with acute vasculitis had no PCV2-specific antibodies (or a low titer of), whereas the pigs with lymphohistiocytic and plasmacytic vasculitis developed high antibody titers against this virus. These observations suggest that (1) acute vasculitis observed in the current studies is directly caused by PCV2b, (2) chronic vasculitis may in part be mediated by the subsequent immune response, and (3) host factors and viral strain may both contribute to vasculitis in animals infected with PCV2b.

Enteric colonization by staphylococcus delphini in four ferret kits with diarrhoea.

Four, 1-to 4-week-old ferret kits were submitted to the Diagnostic Center for Population and Animal Health at Michigan State University for post-mortem examination. Grossly, multiple bowel loops in all ferret kits were distended by mucoid faecal material. Microscopically, there was no evidence of inflammation or notable alteration to the normal mucosal morphology. Gram-positive coccoid bacteria colonized variable segments of the small intestine. These bacteria were identified as Staphylococcus delphini by phenotypic and molecular analyses. Enzyme-linked immunosorbent assay for detection of Staphylococcus enterotoxins was positive and polymerase chain reaction detected the gene for Staphylococcus enterotoxin E in the isolates. The hypersecretory diarrhoea in these ferret kits may have been associated with colonization of the small intestine by S. delphini, cultures of which were shown in vitro to be potentially capable of producing enterotoxin E. The condition described in these ferrets is similar to 'sticky' kit syndrome in mink.

Feasibility of aqueous shunts for reduction of intraocular pressure in horses.

REASONS FOR PERFORMING STUDY: Based on the current literature, neither medical, surgical nor combination therapy adequately controls equine glaucoma for many horses. Aqueous shunts have been useful in other species to control glaucoma. OBJECTIVES: To determine whether aqueous shunts in normal equine eyes significantly reduce intraocular pressure (IOP) without causing vision threatening complications. STUDY DESIGN: Prospective experimental trial. METHODS: Aqueous shunts were placed in 7 normal eyes of 4 horses. The shunts were placed dorsotemporally. Examinations were initially performed daily for 7 days and after that every 3 days through 4 weeks after implantation. Horses were then subjected to euthanasia and globes enucleated for routine histological examination. The IOPs for each day post operatively were compared to the preoperative value (Day -1) using a Wilcoxon signed ranks test. Significance was set at P<0.05. RESULTS: The mean IOP preoperatively (20.7 ± 3.0 mmHg) was significantly higher than on any post operative day (P values ranged from 0.018 to 0.048). The aqueous shunts remained in situ for the entire study. Two eyes developed corneal ulcers that resolved. Shallow anterior chambers were noted in 2 eyes after shunt placement, which normalised after placement of full eye cup masks. Histologically, 7/7 eyes had fibrosis surrounding the implant. Minimal peripheral neovascularisation and neutrophilic keratitis were noted in 5/7 eyes. Corneal damage was scored as none in 3/7, mild in 2/7, moderate in 1/7 and marked in 1/7 eyes. CONCLUSIONS: After placement of aqueous shunts, a significant decrease in IOP was noted from preoperatively (Day -1) to Day 28 despite fibrosis surrounding the implants. No vision threatening complications were noted. POTENTIAL RELEVANCE: Aqueous shunts may represent a feasible therapeutic option for equine glaucoma. The results of this study suggest that further studies in glaucomatous horses would be warranted.

Coccidioidomycosis presenting as a heart base mass in two dogs.

Two atypical cases of canine coccidioidomycosis presenting as heart base masses are described. An echocardiogram performed in one of the two dogs revealed a large mass at the base of the heart and a computed tomography scan showed that the mass compressed the bronchi, left atrium, aorta and pulmonary arteries. A firm, white or pale yellow mass was found at the base of the heart at necropsy examination in both cases. Microscopical examination of the masses revealed severe, chronic, locally extensive granulomatous or pyogranulomatous inflammation with intralesional spherules consistent with Coccidioides spp. The diagnosis was further confirmed by immunohistochemistry and in-situ hybridization. Coccidioides spp. have been reported to cause pericarditis in dogs, but this is the first description of coccidioidomycosis mimicking a heart-based tumour in dogs.

Listeric meningoencephalomyelitis in a cougar (Felis concolor): characterization by histopathologic, immunohistochemical, and molecular methods.

Listeria monocytogenes has been recognized as an important food-borne pathogen in animals. Records of the disease caused by this bacterium in large felids are, however, rare. The nervous form of listeriosis was diagnosed in a 12-year-old male cougar (Felis concolor) with a several-day history of neurologic disease characterized by excess salivation, head pressing, and circling that progressed to recumbency and death. Microscopically, the main alteration in the brain and spinal cord was a variably severe meningoencephalomyelitis composed mainly of mononuclear cell aggregates with fewer neutrophils. L. monocytogenes was isolated from the brain by microbiological culture, and L. monocytogenes antigen was detected in formalin-fixed, paraffin-embedded sections of brain and spinal cord by immunohistochemical analysis. On the basis of the nucleotide sequence of the 16S rRNA gene, the isolated strain was determined to be serotype 1/2a. Food-borne transmission of the bacterium was suspected, but food was not available for testing.

Idiopathic complex polysaccharide storage disease in an abyssinian cat.

A glycogen storage disease affecting primarily the skeletal muscle and, to a lesser degree, the cardiac muscle, spinal cord, and brain was diagnosed in a 10-year-old neutered Abyssinian cat with a 4-year history of paresis progressing to acute paralysis. Microscopically, these tissues contained inclusions that were pale basophilic in hematoxylin and eosin-stained slides, diastase resistant, periodic acid-Schiff positive, and blue-to-almost black with iodine stain. By transmission electron microscopy, the inclusions consisted of cytosolic, usually sharply demarcated, nonmembrane-bound deposits of finely granular and filamentous material. On the basis of the structural and histochemical staining characteristics, the inclusions were believed to be aggregates of abnormally stored, unbranched glycogen. A defect in glucose metabolism is suspected to be the underlying pathologic process, but an exact cause remains elusive.

Extensive lesions of monkeypox in a prairie dog (Cynomys sp).

Monkeypox with extensive lesions was diagnosed in a prairie dog that was involved in a recent human outbreak of monkeypox in the Midwestern United States. Gross lesions included oral ulcers, pulmonary consolidation, enlarged cervical and thoracic lymph nodes, and multifocal, small, white umbilicated plaques in the gastrointestinal wall. Microscopic lesions were extensive in the lungs and consisted of fibrinonecrotic bronchopneumonia with vasculitis and poorly defined eosinophilic intracytoplasmic inclusion bodies in cells thought to be alveolar epithelial cells, histiocytes, and fibroblasts. Multifocal necrotizing lesions, often accompanied by myxedema, were also present in most of the other examined organs. Aggregates of pox viral particles were observed within lesions by transmission electron microscopy. Monkeypox virus infection was confirmed by real-time polymerase chain reaction and virus culture at the Centers for Disease Control and Prevention. This report highlights the difficulties of rapid diagnosis of exotic or emerging diseases and further substantiates the prairie dog as an animal model of monkeypox.