POSTMORTEM COMPUTED TOMOGRAPHY AND MAGNETIC RESONANCE IMAGING FINDINGS IN A CASE OF COINFECTION OF DOLPHIN MORBILLIVIRUS AND ASPERGILLUS FUMIGATUS IN A JUVENILE BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS).

A freshly dead juvenile bottlenose dolphin (Tursiops truncatus), recovered from the waters near Sand Key, Clearwater, FL, was imaged postmortem using computed tomography and magnetic resonance imaging prior to conventional necropsy. The pattern of imaging findings in the brain was compatible with severe multifocal meningoencephalitis with intralesional necrosis and/or hemorrhage, and the pattern of imaging findings in the lungs was compatible with severe multifocal bronchopneumonia. The subsequent investigation included necropsy, histology, culture, and molecular diagnostics and demonstrated disseminated coinfection of dolphin morbillivirus and Aspergillus fumigatus. This is the first report documenting the cross-sectional imaging findings of this important cetacean comorbidity and demonstrates advances in modern, cooperative investigations of marine mammal mortality events.

A RETROSPECTIVE STUDY OF THE LESIONS ASSOCIATED WITH IRON STORAGE DISEASE IN CAPTIVE EGYPTIAN FRUIT BATS (ROUSETTUS AEGYPTIACUS).

Egyptian fruit bats (Rousettus aegyptiacus) are one of many species within zoologic collections that frequently develop iron storage disease. The goals of this retrospective multi-institutional study were to determine the tissue distribution of iron storage in captive adult Egyptian fruit bats and the incidence of intercurrent neoplasia and infection, which may be directly or indirectly related to iron overload. Tissue sections from 83 adult Egyptian fruit bats were histologically evaluated by using tissue sections stained with hematoxylin and eosin, trichrome, and Prussian blue techniques. The liver and spleen consistently had the largest amount of iron, but significant amounts of iron were also detected in the pancreas, kidney, skeletal muscle, and lung. Hepatocellular carcinoma (HCC; 11) was the most common neoplasm, followed by cholangiocarcinoma (4). Extrahepatic neoplasms included bronchioloalveolar adenoma (3), pulmonary carcinosarcoma (1), oral sarcoma (1), renal adenocarcinoma (1), transitional cell carcinoma of the urinary bladder (1), mammary gland adenoma (1), and parathyroid adenoma (1). There were also metastatic neoplasms of undetermined primary origin that included three poorly differentiated carcinomas, a poorly differentiated sarcoma, and a neuroendocrine tumor. Bats with hemochromatosis were significantly more likely to have HCC than bats with hemosiderosis (P = 0.032). Cardiomyopathy was identified in 35/77 bats with evaluable heart tissue, but no direct association was found between cardiac damage and the amount of iron observed within the liver or heart. Hepatic abscesses occurred in multiple bats, although a significant association was not observed between hemochromatosis and bacterial infection. To the authors' knowledge, this is the first publication providing evidence of a positive correlation between hemochromatosis and HCC in any species other than humans.

Characterization of an Avipoxvirus From a Bald Eagle ( Haliaeetus leucocephalus ) Using Novel Consensus PCR Protocols for the rpo147 and DNA-Dependent DNA Polymerase Genes.

A juvenile female bald eagle ( Haliaeetus leucocephalus ) was presented with emaciation and proliferative periocular lesions. The eagle did not respond to supportive therapy and was euthanatized. Histopathologic examination of the skin lesions revealed plaques of marked epidermal hyperplasia parakeratosis, marked acanthosis and spongiosis, and eosinophilic intracytoplasmic inclusion bodies. Novel polymerase chain reaction (PCR) assays were done to amplify and sequence DNA polymerase and rpo147 genes. The 4b gene was also analyzed by a previously developed assay. Bayesian and maximum likelihood phylogenetic analyses of the obtained sequences found it to be poxvirus of the genus Avipoxvirus and clustered with other raptor isolates. Better phylogenetic resolution was found in rpo147 rather than the commonly used DNA polymerase. The novel consensus rpo147 PCR assay will create more accurate phylogenic trees and allow better insight into poxvirus history.

RENAL AND CLOACAL CRYPTOSPORIDIOSIS (CRYPTOSPORIDIUM AVIAN GENOTYPE V) IN A MAJOR MITCHELL'S COCKATOO (LOPHOCHROA LEADBEATERI).

A 7-yr-old male Major Mitchell's cockatoo (Lophochroa leadbeateri) presented with a recent history of lethargy and anorexia. Physical examination revealed poor body condition and cloacal prolapse. Abnormalities on serum chemistry included severe hyperuricemia and hyperphosphatemia with a low calcium-to-phosphorus ratio. Symptomatic treatment was initiated including intravenous fluids and antibiotics. The bird continued to decline and died within a few days. Visceral gout and renal and cloacal pathology were observed on gross necropsy. Histopathology revealed chronic inflammation within the kidney, ureter, and cloaca in association with protozoal organisms and an invasive cloacal adenocarcinoma tumor. The location and morphology was consistent with Cryptosporidium sp., confirmed by immunohistochemistry and molecular testing. Direct sequencing identified Cryptosporidium avian genotype V. To the author's knowledge, this is the first reported infection of Cryptosporidium avian genotype V associated with clinical disease in birds and the first renal Cryptosporidium infection in a psittacine.

Medium-grade astrocytoma in a cougar (Puma concolor).

A 17-year-old, male castrated cougar (Puma concolor) was presented minimally responsive and severely depressed, with bilateral mydriasis and absent pupillary light response. On gross examination of the brain, there was a tan-to-gray, invasive mass with a central cavitation on the ventral aspect in the left cerebral hemisphere, rostral to the caudate nucleus. On histopathologic examination, the mass was composed of sheets of medium-sized, round-to-polygonal cells that were multifocally separated by islands of neuropil. Approximately 80% of the neoplastic cells showed strong cytoplasmic labeling for glial fibrillary acidic protein. These findings were consistent with a medium-grade astrocytoma. To the authors' knowledge, neoplastic disease of the central nervous system has not been previously reported in cougars.

Diabetogenic effect of a series of tricyclic delta opioid agonists structurally related to cyproheptadine.

The unexpected observation of a hyperglycemic effect of some tricycle-based delta opioid receptor (DOR) agonists led to a series of studies to better understand the finding. Single administration of two novel tricyclic DOR agonists dose dependently elevated rat plasma glucose levels; 4-week toxicology studies confirmed the hyperglycemic finding and further revealed pancreatic ß-cell hypertrophy, including vacuole formation, as well as bone dysplasia and Harderian gland degeneration with regeneration. Similar diabetogenic effects were observed in dog. A review of the literature on the antiserotonergic and antihistaminergic drug cyproheptadine (CPH) and its metabolites revealed shared structural features as well as similar hyperglycemic effects to the present series of DOR agonists. To further evaluate these effects, we established an assay measuring insulin levels in the rat pancreatic ß-cell-derived RINm5F cell line, extensively used to study CPH and its metabolites. Like CPH, the initial DOR agonists studied reduced RINm5F cell insulin levels in a concentration-dependent manner. Importantly, compound DOR potency did not correlate with the insulin-reducing potency. Furthermore, the RINm5F cell insulin results correlated with the diabetogenic effect of the compounds in a 5-day mouse study. The RINm5F cell insulin assay enabled the identification of aryl-aryl-amine DOR agonists that lacked an insulin-reducing effect and did not elevate blood glucose in repeated dosing studies conducted over a suprapharmacologic dose range. Thus, not only did the RINm5F cell assay open a path for the further discovery of DOR agonists lacking diabetogenic potential but also it established a reliable, economical, and high-throughput screen for such potential, regardless of chemotype or target pharmacology. The present findings also suggest a mechanistic link between the toxicity observed here and that underlying Wolcott-Rallison Syndrome.

Evaluation of felbamate and other antiepileptic drug toxicity potential based on hepatic protein covalent binding and gene expression.

Felbamate is an antiepileptic drug that is associated with minimal toxicity in preclinical species such as rat and dog but has an unacceptable incidence of serious idiosyncratic reactions in man. Idiosyncratic reactions account for over half of toxicity-related drug failures in the marketplace, and improving the preclinical detection of idiosyncratic toxicities is thus of paramount importance to the pharmaceutical industry. The formation of reactive metabolites is common among most drugs associated with idiosyncratic drug reactions and may cause deleterious effects through covalent binding and/or oxidative stress. In the present study, felbamate was compared to several other antiepileptic drugs (valproic acid, carbamazepine, phenobarbital, and phenytoin), using covalent binding of radiolabeled drugs and hepatic gene expression responses to evaluate oxidative stress/reactive metabolite potential. Despite causing only very mild effects on covalent binding parameters, felbamate produced robust effects on a previously established oxidative stress/reactive metabolite gene expression signature. The other antiepileptic drugs and acetaminophen are known hepatotoxicants at high doses in the rat, and all increased covalent binding to liver proteins in vivo and/or to liver microsomes from human and rat. With the exception of acetaminophen, valproic acid exhibited the highest covalent binding in vivo, whereas carbamazepine exhibited the highest levels in vitro. Pronounced effects on oxidative stress/reactive metabolite-responsive gene expression were observed after carbamazepine, phenobarbital, and phenytoin administration. Valproic acid had only minor effects on the oxidative stress/reactive metabolite indicator genes. The relative ease of detection of felbamate based on gene expression results in rat liver as having potential oxidative stressor/reactive metabolites indicates that this approach may be useful in screening for potential idiosyncratic toxicity. Together, measurements of gene expression along with covalent binding should improve the safety assessment of candidate drugs.