Influence of genetic background on tumor karyotypes: evidence for breed-associated cytogenetic aberrations in canine appendicular osteosarcoma.

Recurrent chromosomal aberrations in solid tumors can reveal the genetic pathways involved in the evolution of a malignancy and in some cases predict biological behavior. However, the role of individual genetic backgrounds in shaping karyotypes of sporadic tumors is unknown. The genetic structure of purebred dog breeds, coupled with their susceptibility to spontaneous cancers, provides a robust model with which to address this question. We tested the hypothesis that there is an association between breed and the distribution of genomic copy number imbalances in naturally occurring canine tumors through assessment of a cohort of Golden Retrievers and Rottweilers diagnosed with spontaneous appendicular osteosarcoma. Our findings reveal significant correlations between breed and tumor karyotypes that are independent of gender, age at diagnosis, and histological classification. These data indicate for the first time that individual genetic backgrounds, as defined by breed in dogs, influence tumor karyotypes in a cancer with extensive genomic instability.

Assessment of novel avian renal disease markers for the detection of experimental nephrotoxicosis in pigeons (Columba livia).

Renal disease is a major cause of illness in captive and wild avian species. Current renal disease markers (e.g., uric acid, blood urea nitrogen, and creatinine) are insensitive. Two endogenous markers, creatine and N-acetyl-beta-D-glucosaminidase (NAG), were selected for study in the pigeon (Columba livia). Representative organs from four pigeons were surveyed to determine those exhibiting the highest level of each marker. In a separate study, NAG and creatine from plasma and urine were assayed before and after gentamicin (50 mg/kg twice daily), administration for up to 9 days. Observer-blinded pathologic scoring (five saline solution controls, 17 treated birds) was used to verify the presence of renal disease that corresponded to marker increases. The first study revealed that kidney tissue had the highest NAG activity (by approximately six times), and pectoral muscle had the most creatine (>900 times). In response to gentamicin, plasma creatine (>five times) and NAG increased (approximately six times), which paralleled uric acid (>10 times). Urine creatine (approximately 60 times) and NAG increased dramatically (approximately 50 times) in response to gentamicin. In conclusion, NAG, especially in the urine, may be of value to noninvasively detect renal toxin exposures and to monitor potentially nephrotoxic drugs, and might be of value to screen free-ranging birds in large exhibits or in the wild by assaying fresh urate samples at feeding stations.

Enhancing antimelanoma immune responses through apoptosis.

We examined the feasibility of using tumor apoptosis at accessible sites to enhance antimelanoma immune responses in a model of spontaneous canine melanoma. We show that priming peripheral blood mononuclear cells with apoptotic melanoma cells significantly enhanced autologous and allogeneic lymphokine-activated killing of tumor cells. Since various pathways required for intrinsic apoptosis are often inactivated in melanoma, we used Fas ligand (FasL) overexpression to promote extrinsic apoptosis. FasL induced apoptosis in five of six cell lines. Each of the susceptible lines, but not the resistant one, expressed Fas mRNA. In addition, direct intratumoral administration of FasL DNA to tumor-bearing dogs was safe, with no adverse events reported over 7 days of observation. A reduction of tumor burden was seen in three of five dogs treated. The reduction of tumor volume was correlated with Fas expression by the tumors, although one dog with a Fas-negative tumor survived for 82 weeks after treatment. Our data show that overexpression of FasL is suitable to promote apoptosis of Fas(+) melanomas, and support the notion that priming immune responder cells with apoptotic tumor cells may enhance antitumor responses. The results also suggest that intratumoral administration of FasL offers a safe route for therapeutic gene delivery.

High Fungal Spore Load in Corncob Bedding Associated with Fungal-Induced Rhinitis in Two Rats.

Two types of commonly used contact bedding, aspen chip and corncob, were analyzed for the presence of fungal spores after two rats in a toxicology study were diagnosed with fungal rhinitis. A range of 700 to 5440 spores per g consisting of six species of fungus (Cladosporidium, Acremonium, Penicillium, Aspergillus, Fusarium, and Scolobasidium) were isolated from non-autoclaved corncob bedding obtained from two vendors. Negligible spore counts were detected in autoclaved corncob bedding and from either autoclaved or non-autoclaved aspen chip bedding. The present study demonstrates that high fungal spore counts may occur in untreated corncob bedding and that these may be associated with opportunistic disease in healthy, non-immunosuppressed animals. Autoclaving reduced the viable fungal spore load to an undetectable level.

Myasthenia gravis in a polar bear (Ursus maritimus).

A 14.6-yr-old, female, multiparous polar bear (Ursus maritimus) acutely developed an apparent hind limb weakness. Physical examination and diagnostic tests including a hemogram, serum biochemistry, electrolytes, radiographs, and myelogram did not provide a definitive diagnosis. No improvement in condition was noted during 4 days of supportive care, and the bear was euthanized. An ovoid mass was present in the anterior mediastinum, and a thymoma was confirmed histologically. Compared with control polar bears, elevated serum acetylcholine receptor (AChR) antibodies (0.13 +/- 0.06 nmol/L vs. 0.86 nmol/L) were detected by immunoprecipitation radioimmunoassay, which is consistent with myasthenia gravis (MG) in other species. Although the AChR antibody test has not been validated in the polar bear, we are confident in the postmortem diagnosis of MG, which is commonly associated with thymoma in other species.

Toxoplasmosis in Pallas' cats (Otocolobus felis manul) at the Denver Zoological Gardens.

In May 1996 the Denver Zoological Gardens obtained two male and two female Pallas' cats (Otocolobus felis manul) that were wild-caught in the Ukraine. These animals were part of a group of 16 wild-caught adults (eight male and eight female) imported to the United States and Canada between 1995 and 1996. The Denver Zoological Gardens cats were quarantined at the zoo hospital for approximately I mo. During the quarantine period they were immobilized for physical examination, and sera were obtained from them to evaluate for exposure to Toxoplasma gondii. All cats were positive for T. gondii antibodies by latex agglutination (titers from 1:512 to 1:1,024). After being paired for breeding, one pair produced two litters, and another pair produced four litters, a total of 17 kittens between 1997 and 2001. Four kittens and two young adults died from a disseminated granulomatous and necrotizing inflammation consistent with toxoplasmosis. Toxoplasma gondii infection was confirmed in all six deceased cats by polymerase chain reaction performed on formalin-fixed tissues. An additional five kittens disappeared and were not available for necropsy. The fatality rate from toxoplasmosis was 35.3% (6/17) for cats that were available for necropsy and could have been as high as 64.7% (11/17) if it were assumed that the disappeared kittens were also affected. The Pallas' kitten survival rate at the Denver Zoological Gardens was 35.3%. This article describes the clinical and pathologic features of toxoplasmosis in a group of Pallas' cats at the Denver Zoological Gardens.

Novel poxvirus infection in northern and southern sea otters (Enhydra lutris kenyoni and Enhydra lutris neiris), Alaska and California, USA.

Small superficially ulcerated skin lesions were observed between October 2009 and September 2011 during captive care of two orphaned sea otter pups: one northern (Enhydra lutris kenyoni) in Alaska and one southern (Enhydra lutris nereis) in California. Inclusions consistent with poxviral infection were diagnosed by histopathology in both cases. Virions consistent with poxvirus virions were seen on electron microscopy in the northern sea otter, and the virus was successfully propagated in cell culture. DNA extraction, pan-chordopoxviral PCR amplification, and sequencing of the DNA-dependent DNA polymerase gene revealed that both cases were caused by a novel AT-rich poxvirus. Bayesian and maximum likelihood phylogenetic analyses found that the virus is divergent from other known poxviruses at a level consistent with a novel genus. These cases were self-limiting and did not appear to be associated with systemic illness. To our knowledge, this is the first report of a poxvirus in a mustelid species. The source of this virus, mode of transmission, zoonotic potential, and biological significance are undetermined.

Molecular subtypes of osteosarcoma identified by reducing tumor heterogeneity through an interspecies comparative approach.

The heterogeneous and chaotic nature of osteosarcoma has confounded accurate molecular classification, prognosis, and prediction for this tumor. The occurrence of spontaneous osteosarcoma is largely confined to humans and dogs. While the clinical features are remarkably similar in both species, the organization of dogs into defined breeds provides a more homogeneous genetic background that may increase the likelihood to uncover molecular subtypes for this complex disease. We thus hypothesized that molecular profiles derived from canine osteosarcoma would aid in molecular subclassification of this disease when applied to humans. To test the hypothesis, we performed genome wide gene expression profiling in a cohort of dogs with osteosarcoma, primarily from high-risk breeds. To further reduce inter-sample heterogeneity, we assessed tumor-intrinsic properties through use of an extensive panel of osteosarcoma-derived cell lines. We observed strong differential gene expression that segregated samples into two groups with differential survival probabilities. Groupings were characterized by the inversely correlated expression of genes associated with 'G2/M transition and DNA damage checkpoint' and 'microenvironment-interaction' categories. This signature was preserved in data from whole tumor samples of three independent dog osteosarcoma cohorts, with stratification into the two expected groups. Significantly, this restricted signature partially overlapped a previously defined, predictive signature for soft tissue sarcomas, and it unmasked orthologous molecular subtypes and their corresponding natural histories in five independent data sets from human patients with osteosarcoma. Our results indicate that the narrower genetic diversity of dogs can be utilized to group complex human osteosarcoma into biologically and clinically relevant molecular subtypes. This in turn may enhance prognosis and prediction, and identify relevant therapeutic targets.

Cytology of canine malignant histiocytosis.

Cytologic features of bone marrow, tissue, and abdominal fluid in seven cases of malignant histiocytosis in dogs are described, and histopathology, hematology, and serum biochemistry of the cases are reviewed. Diagnosis of malignant histiocytosis was confirmed by tissue morphology and immunohistochemistry; neoplastic cells in all cases had positive immunoreactivity to lysozyme. This stain can be used to definitively establish the diagnosis of malignant histiocytosis on cytology specimens as well as tissue sections. Cytologic findings included numerous pleomorphic, large, discrete mononuclear cells with abundant, lightly basophilic, vacuolated, granular cytoplasm. Nuclei were round to oval to reniform with marked anisocytosis and anisokaryosis; nucleoli were prominent. Mitotic figures, often bizarre, were occasionally seen. Multinucleated giant cells and phagocytosis of erythrocytes and leukocytes were prominent features in cytologic preparations in four cases. Four dogs were anemic, five dogs were thrombocytopenic, and three dogs were hypercalcemic. Breeds affected included Doberman Pinscher (1), Golden Retriever (2), Flat Coated Retriever (3), and mixed-breed dog (1).

Canine malignant hemangiosarcoma as a model of primitive angiogenic endothelium.

Hemangiosarcoma (HSA) is a common untreatable cancer of dogs that resembles human angiosarcoma. Detailed studies of these diseases have been historically hindered by the paucity of suitable reagents. Here, we show that expression of CD117 (c-Kit) can distinguish primitive (malignant) from mature (benign) proliferative endothelial lesions, and we describe eight independent cell lines derived from canine HSA explants. Endothelial origin was confirmed by sustained expression of surface CD105 (endoglin), CD146 (MUC18), and CD51/CD61 (alpha(v)beta(3) integrin). The cell lines showed anchorage-independent growth and were motile and invasive when cultured on a basement membrane matrix. They required endothelial growth factors for growth and survival, and they could be induced to form tubular structures resembling blood vessels when cultured under low calcium conditions. The formation of vessel-like structures was blocked by nicotine, and restored by FK506, suggesting that 'nuclear factor of activated T cells' activity prevents differentiation of these cells. In summary, these cell lines represent a unique and novel resource to improve our understanding of endothelial cell biology in general and canine HSA in particular.