Histologic, immunohistochemical, and electron microscopic features of a unique pulmonary tumor in cockatiels (Nymphicus hollandicus): six cases.

A unique form of pulmonary malignancy develops in cockatiels. This report describes the gross, histologic, electron microscopic, and immunohistochemical features of this tumor in 6 cockatiels. DNA in-situ hybridization for polyomavirus in the neoplasm was also performed. The tumor was comprised predominantly of compact sheets of anaplastic round to polygonal cells. All tumors had a high mitotic index, and had occasional large clear to slightly basophilic intranuclear inclusions that caused peripheral dispersal or complete masking of chromatin. Tumors were invasive but convincing metastases were not observed. Transmission electron microscopy identified intracytoplasmic intermediate filaments, desmosomes between cells, and intranuclear cytoplasmic invaginations corresponding to the intranuclear inclusions in light microscopic sections. Neoplastic cells stained positive for vimentin, lysozyme, and in 1 bird, pan cytokeratin. All 6 pulmonary neoplasms were negative for avian polyomavirus using the FN-19/FN-40 cocktail and the long VP-1 probe. We propose that these tumors may be poorly differentiated carcinomas of pulmonary or thymic origin.

Meningoencephalitis in a polar bear caused by equine herpesvirus 9 (EHV-9).

A 12-year-old female polar bear (Ursus maritimus) developed a sudden onset of muscle tremors, erratic circling, increased blinking, head shaking, and ptyalism, which progressed to partial and generalized seizures. Ancillary diagnostic tests were inconclusive, and the only significant laboratory finding was nonsuppurative pleocytosis of cerebrospinal fluid. Euthanasia was elected. Microscopic evaluation demonstrated multifocal, random nonsuppurative meningoencephalitis involving most prominently the rostral cerebral cortex, as well as the thalamus, midbrain, and rostral medulla. Lesions consisted of inflammation, neuronal necrosis, gliosis, and both neuronal and glial basophilic intranuclear inclusion bodies. Immunohistochemistry with a polyclonal antibody reactive to several equine herpesviruses was positive within affected areas of the brain, and polymerase chain reaction conclusively demonstrated the presence of only equine herpesvirus 9. The clinical and morphologic features of this case resemble other fatal herpesvirus encephalitides derived from interspecies transmission and underscore the need for extreme caution when managing wild or captive equids.

Cluster of cases of malignant schwannoma in cattle.

Between 1998 and 2001, several cases of ataxia and paresis followed by recumbency and death were reported in cows from different farms in a restricted area of the Argentinian Patagonia. Five cases of this cluster were studied and a diagnosis of malignant schwannoma was established. Electron microscopy (em) of tumour samples from three of the animals revealed intracytoplasmic or interstitial structures resembling retroviral particles. Attempts to isolate a viral agent from the tumours were unsuccessful but the epidemiological data and the em findings suggest a viral aetiology.

Formation of phospholipid-rich HDL: a model for square-packing lipoprotein particles found in interstitial fluid and in abetalipoproteinemic plasma.

The major bovine HDL subfraction, fraction I-HDL, was incubated with increasing amounts of dimyristoylphosphatidylcholine (DMPC). HDL size, as determined by gradient gel electrophoresis and electron microscopy, increased with increasing HDL-phospholipid to DMPC mole ratios. Control fraction I-HDL were spherical, hexagonally-packing particles with a peak on gradient gel electrophoresis at 12.3 +/- 0.1 nm; at a ratio of 1:0.5, larger, mainly spherical particles with a peak at 12.9 +/- 0.08 nm were formed. At a ratio of 1:1, occasional square-shaped particles were seen by electron microscopy; by gradient gel analysis, the mean diameter of the HDL-product increased to 13.7 +/- 0.1 nm. At the 1:2 ratio, extensive domains of square-packing particles were noted; the major size peak of this product was 14.6 +/- 0.08 nm. In all incubations with DMPC, a small 9.4 +/- 0.08 nm product was formed; it was most pronounced at the 1:2 ratio. The large, less dense particles generated by incubation contained apolipoprotein A-I and small molecular weight proteins. The 9.4 nm product contained only apolipoprotein A-I. The less dense product formed during incubation at the 1:2 ratio had a decreased protein-to-lipid ratio relative to control HDL and a 2-fold increase in percent phospholipid. At a 1:2 ratio, incorporation of DMPC into fraction I-HDL results in the loss of one molecule of apolipoprotein A-I; the resultant particle is a stable phospholipid-rich and protein-poor HDL which has a square-packing geometry. These phospholipid-laden HDL are morphologically similar to lipoproteins isolated from interstitial fluid or from plasma of abetalipoproteinemic patients. Our data suggest that the unusual morphological properties of the latter biologically formed particles may be due to increases in the polar lipid contents, and concomitant decreases in surface protein.

Physical and chemical characteristics of apolipoprotein A-I-lipid complexes produced by Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene.

Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene linked to the human metallothionein gene promoter region secrete large quantities of apolipoprotein A-I (7.1 +/- 0.4% total secreted protein) in the presence of zinc. Approx. 16% of the secreted apolipoprotein A-I is complexed with lipid and can be isolated ultracentrifugally at d less than or equal to 1.21 g/ml. The latter complexes are composed of discs and vesicles as judged by electron microscopy and can be further separated by column chromatography into three fractions: fraction I, mostly vesicles (60-260 nm) and large discs (18-20 nm diameter); fraction II, discs 14.2 +/- 2.6 nm diameter; and fraction III, nonresolvable by electron microscopy. The latter fraction is extremely lipid-poor (94% protein, 6% phospholipid); in contrast, the protein, phospholipid and unesterified cholesterol content for the other fractions are 43, 33 and 24%, respectively, for fraction I and 53, 33 and 14%, respectively, for fraction II. Fraction II particles contain three and four apolipoprotein A-Is per particle as determined by protein crosslinking while large structures in fraction I contain primarily six to seven apolipoprotein A-Is per particle. Following incubation with purified lecithin: cholesterol acyltransferase, discoidal particles were transformed into apparent spherical particles 12.9 +/- 3.4 nm diameter; this transformation coincided with 19-21% conversion of unesterified cholesterol to esterified cholesterol. The apolipoprotein A-I-lipid complexes isolated from Chinese hamster ovary cell media are similar to nascent HDL found in plasma of lecithin:cholesterol acyltransferase-deficient patients and those secreted by the human hepatoma line, Hep G2. The ability of the Chinese hamster ovary cell nascent HDL-like particles to undergo transformation in the presence of purified lecithin:cholesterol acyltransferase indicates that they are functional particles.

Dissociation of apolipoprotein A-I from porcine and bovine high density lipoproteins by guanidine hydrochloride.

Dissociation of apolipoprotein A-I from pig and steer high density lipoproteins (HDL) deficient in apoA-II was determined by exposing native HDL fractions to 6 M guanidine hydrochloride (Gdn-HCl) at 37 degrees C for periods from 5 min to 18 h. Bovine high density lipoprotein (HDL-B) was isolated at d 1.063--1.100 g/ml while porcine high density lipoprotein (HDL-P) was isolated at d 1.125--1.21 g/ml. Incubation for 5 min with Gdn-HCl resulted in a 45 and 3% loss of apo-A-I from HDL-P and HDL-B, respectively. Exposure to the denaturant for 3 h resulted in a 75% loss of apoA-I from HDL-P and a 30% loss from HDL-B. Analytic ultracentrifugation, patterns paralleled the degree of apoA-I dissociation from each HDL species. The initial flotation peak for HDL-P shifted from F degrees 1.20 2.68 to F degrees 1.20 10.75 after 3 h exposure while HDL-B showed only a small shift from F degrees 1.20 8.30 to F degrees 1.20 8.96 after 3 h exposure. HDL-P particle diameter increased 25% after 5 min of Gdn-HCl treatment and large, flattened structures predominated after 3 h. There was no changes in the size of HDL-B after 5 min exposure and only 16% increase in particle diameter after 3 h. The difference in behavior of HDL-B and HDL-P to Gdn-HCl exposure is discussed in terms of differences in apolipoprotein A-I amino acid composition, interaction of apolipoprotein A-I with phospholipids and the possible involvement of the cholesteryl ester core.

Morphological and molecular characterization of Encephalitozoon hellem in hummingbirds.

Microsporidiosis was identified as a cause of enteritis in wild, migratory hummingbirds (Calypte anna). Electron microscopic examinations of parasites showed microsporidian spores with a double spore coat and a polar filament containing four to six coils, compatible with the genus Encephalitozoon. Molecular analysis of ribosomal RNA genes further identified the parasites from droppings and small intestinal segments as Encephalitozoon hellem, genotype I. Microsporidial spores were identified in 19% of droppings from C. anna, Archilochus alexandri and Selasporus sasin using Gram or modified trichrome staining methods. Since E. hellem is an opportunistic pathogen in immunocompromised humans, the pathogenic potential in avian hosts, the zoonotic potential of this parasite, and the role of birds as reservoirs needs to be further explored.

Spirochetes isolated from dairy cattle with papillomatous digital dermatitis and interdigital dermatitis.

Two groups of spirochetes were isolated from papillomatous digital dermatitis (PDD) lesions in dairy cattle. The two groups could be readily differentiated on the basis of morphologic and immunologic characteristics and enzymatic activity. A spirochete isolated from an interdigital dermatitis (IDD) lesion appeared morphologically and antigenically similar to spirochetes in one of the PDD groups and exhibited an identical enzyme activity pattern. The two groups of PDD spirochetes had characteristics most consistent with the genus Treponema. The PDD and IDD isolates differed morphologically from previously described bovine Treponema spp. Although spirochetes have been observed to be one of the predominant bacterial morphotypes in PDD and IDD and are found invading the stratum spinosum and dermal papillae in PDD lesions, the significance of these spirochetes in the etiopathogenesis of PDD and IDD is presently unknown.

Four-hour processing of clinical/diagnostic specimens for electron microscopy using microwave technique.

A protocol for routine 4-hour microwave tissue processing of clinical or other samples for electron microscopy was developed. Specimens are processed by using a temperature-restrictive probe that can be set to automatically cycle the magnetron to maintain any designated temperature restriction (temperature maximum). In addition, specimen processing during fixation is performed in 1.7-ml microcentrifuge tubes followed by subsequent processing in flow-through baskets. Quality control is made possible during each step through the addition of an RS232 port to the microwave, allowing direct connection of the microwave oven to any personal computer. The software provided with the temperature probe enables the user to monitor time and temperature on a real-time basis. Tissue specimens, goat placenta, mouse liver, mouse kidney, and deer esophagus were processed by conventional and microwave techniques in this study. In all instances, the results for the microwave-processed samples were equal to or better than those achieved by routine processing techniques.

A disease resembling inclusion body disease of boid snakes in captive palm vipers (Bothriechis marchi).

Between April 1998 and June 1999, 8 palm vipers (Bothriechis marchi) were diagnosed with a disease similar to inclusion body disease (IBD) of boids. Six palm vipers were captive bred, and 2 were wild caught. All of the vipers were adults at the time of death. Three palm vipers were found dead with no premonitory clinical signs, and 5 had anorexia plus possibly 1 of the following clinical signs: regurgitation, paresis, and dehydration. Histologically, all snakes had intracytoplasmic, round to oval, single to multiple eosinophilic inclusion bodies in hepatocytes and renal tubular epithelial cells. Inclusion bodies were distributed among other organs with varying frequency. Common concurrent histologic lesions were urate nephrosis, septic thrombi, and hepatocellular degeneration. Ultrastructurally, inclusions had features similar to inclusions in boid snakes with IBD.

Comparison of the 5.8S rRNA gene and internal transcribed spacer regions of trichomonadid protozoa recovered from the bovine preputial cavity.

Sequence analysis of the 5.8S rRNA gene and the internal transcribed spacer regions (ITSRs) was used to compare trichomonadid protozoa (n = 39) of varying morphologies isolated from the bovine preputial cavity. A multiple sequence alignment was performed with bovine isolate sequences and other trichomonadid protozoa sequences available in GenBank. As a group, Tritrichomonasfoetus isolates (n = 7) had nearly complete homology. A similarity matrix showed low homology between the T. foetus isolates and other trichomonads recovered from cattle (<70%). Two clusters of trichomonads other than T. foetus were identified. Eighteen isolates comprised 1 group. These isolates shared >99% homology among themselves and with Pentatrichomonas hominis. The other non-T. foetus cluster (n = 14) did not exhibit a high degree of homology (<87%) with other bovine isolates or any of the trichomonad sequences available in GenBank. The sequence homology among isolates in that cluster was >99%, except for 1 isolate that varied from the others in both ITSRs (approximately 2% dissimilarity). Sequence analysis of the 5.8S rRNA gene and ITSRs was useful for comparing trichomonadid protozoa isolated from the bovine preputial cavity and demonstrated that 2 distinct types of trichomonads constituted the non-T. foetus isolates recovered from the bovine preputial cavity.

A retrospective study of circovirus infection in pigeons: nine cases (1986-1993).

Circovirus infections were diagnosed in 12 pigeons from the United States 4 pigeons from Australia, and 1 pigeon from Canada (1986-1993). Circovirus was identified by electron microscopic examination of basophilic botryoid cytoplasmic inclusions that had a histologic appearance similar to that of psittacine beak and feather disease virus inclusions. Inclusions were seen in splenic, bursal, gut-associated, and bronchus-associated lymphoid tissue macrophages and in bursal epithelial cells. Inclusions were composed of paracrystalline arrays of tightly packed, nonenveloped icosahedral virions 14-17 nm in diameter. Histologic changes in the spleens ranged from lymphofollicular hyperplasia with mild discrete lymphocellular necrosis to lymphoid depletion and diffuse histiocytosis. Lesions in the bursa of Fabricius ranged from mild lymphocellular necrosis to severe cystic bursal atrophy. Remaining histologic findings coincided with concurrent bacterial, viral, fungal, and parasitic infections. Immunoperoxidase staining and DNA in situ hybridization demonstrated that pigeon circovirus is distinct from psittacine beak and feather disease virus; however both viruses apparently share some homologous DNA sequences. Clinical and diagnostic findings indicate that pigeon circovirus may be similar to psittacine beak and feather disease virus with respect to acquired immunodeficiency and subsequent multiple secondary infections.

Particles resembling circovirus in the bursa of Fabricius of pigeons.

Histological examination of the bursae from 12 pigeons under 4 months old revealed basophilic globular inclusion bodies, 5 to 25 microns in diameter, in the cytoplasm and the nuclei of the various bursal follicular cells. Electron microscopy of these inclusions revealed large electron-dense areas containing non-enveloped icosahedral viral particles, 14-19 nm in diameter, either loosely arranged or in paracrystalline array. Similar basophilic globular inclusion bodies were seen in the spleen and cecal tonsils of a few pigeons and in the duodenum of one pigeon. There were various degrees of lymphoid depletion in the bursa, spleen, and bone marrow. The morphology of the inclusions in the bursa and size of the viral particles are most consistent with circovirus. Preliminary studies on the bursae of two pigeons were negative for psittacine beak and feather disease (PBFD) viral antigen and nucleic acid by immunoperoxidase staining, DNA in situ hybridization, and polymerase chain reaction techniques, suggesting that this virus differs from PBFD virus. Most of the pigeons had concurrent infections such as paramyxovirus-1, salmonellosis, herpesvirus, and hepatic and cerebral trichomoniasis associated with adenovirus.

Isolation of a poxvirus from a black-tailed deer (Odocoileus hemionus columbianus).

A poxvirus was isolated during the latter half of 1993 from a black-tailed deer (Odocoileus hemionus columbianus) that died of fulminant adenovirus infection in California (USA). The poxvirus was isolated from a pooled tissue homogenate, after repeated serial blind passages in primary black-tailed deer testicular cells. Based on electron microscopic examination of the virus, we observed morphologic features typical of the genus Orthopoxvirus, although definitive characterization was not done.

Experimental adenovirus hemorrhagic disease in white-tailed deer fawns.

Infection with a newly described endotheliotropic adenovirus was the cause of a 1993 epizootic reminiscent of hemorrhagic disease in California mule deer (Odocoileus hemionus columbianus and O. hemionus hemionus). Pulmonary edema and intestinal luminal hemorrhage, or necrotizing stomatitis associated with systemic or localized vasculitis, respectively, were common lesions seen in animals that died during the epizootic. In order to determine if white-tailed deer (Odocoileus virginianus) also are susceptible to infection and fatal disease with the deer adenovirus, eight white-tailed deer fawns (4- to 6-mo-old) were inoculated with purified deer adenovirus. Four were inoculated intravenously and four were inoculated through the mucous membranes. Seven days post-inoculation, one of the fawns inoculated intravenously died. Pulmonary edema and hemorrhagic enteropathy were associated with pulmonary and intestinal vasculitis with systemic multiorgan distribution of endotheliotropic adenovirus as demonstrated by transmission electron microscopy and immunohistochemistry. Adenovirus was reisolated from lung homogenates of the fawn that died of adenovirus hemorrhagic disease.

Experimental adenovirus hemorrhagic disease in yearling black-tailed deer.

An apparently novel adenovirus was associated with an epizootic of hemorrhagic disease that is believed to have killed thousands of mule deer (Odocoileus hemionus) in California (USA) during 1993-1994. A systemic vasculitis with pulmonary edema and hemorrhagic enteropathy or a localized vasculitis associated with necrotizing stomatitis/pharyngitis/glossitis or osteomyelitis of the jaw were common necropsy findings in animals that died during this epizootic. Six black-tailed yearling deer (O. hemionus columbianus) were inoculated with purified adenovirus isolated from a black-tailed fawn that died of acute adenovirus hemorrhagic disease during the epizootic. Three of six inoculated deer also received intramuscular injections of dexamethasone sodium phosphate every 3 days during the study. Eight days post-inoculation, one deer (without dexamethasone) developed bloody diarrhea and died. Necropsy and histopathologic findings were identical to lesions in free-ranging animals that died of the natural disease. Hemorrhagic enteropathy and pulmonary edema were the significant necropsy findings and there was microscopic vascular damage and endothelial intranuclear inclusion bodies in the vessels of the intestines and lungs. Adenovirus was identified in necrotic endothelial cells in the lungs by fluorescent antibody staining, immunohistochemistry and by transmission electron microscopy. Adenovirus was reisolated from tissues of the animal that died of experimental adenovirus hemorrhagic disease. Similar gross and microscopic lesions were absent in four of six adenovirus-inoculated deer and in the negative control animal which were necropsied at variable intervals during the 14 wk study. One deer was inoculated with purified adenovirus a second time, 12 wk after the first inoculation. Fifteen days after the second inoculation, this deer developed severe ulceration of the tongue, pharynx and rumen and necrotizing osteomyelitis of the mandible which was associated with vasculitis and thrombosis of adjacent large vessels and endothelial intranuclear inclusions. Transmission electron microscopy demonstrated adenovirus within the nuclei of vascular cells and immunohistochemistry demonstrated adenovirus antigen within tonsilar epithelium and in rare vessels.

A Herpesvirus Associated with Mass Mortality of Juvenile and Adult Koi, a Strain of Common Carp.

A herpesvirus was isolated from adult koi, a strain of common carp Cyprinus carpio, suffering mass mortality in two outbreaks-one in the mid-Atlantic region of the United States and the second in Israel. The principal external signs of dying fish were pale and irregularly colored gills. There were few consistent internal signs in either outbreak. The most prominent microscopic lesions were in the gills, where hyperplasia and necrosis of the epithelium were severe. Other lesions included interstitial nephritis, splenitis, and enteritis. Affected cells often contained nuclei with marginated chromatin and faint intranuclear inclusions. Typical herpesvirus particles were present in branchial epithelial cells, hepatocytes, and among circulating leukocytes. Inoculations of the koi fin (KF-1) cell line with tissue extracts from the gill and kidney-spleen resulted in cytopathic effects characterized by severe vacuolation first detected after 7 d incubation at 20°C. Exposures of adult koi to the herpesvirus as propagated in KF-1 cells by bath or intraperitoneal injections resulted in 80-100% mortality during a 26-d period, and the virus was reisolated from the gill, kidney, liver, spleen, intestine, and brain of dead fish. The viral agents from koi in Israel and the United States appear to be similar if not identical; both could be distinguished from Herpesvirus cyprini by indirect fluorescent antibody tests with rabbit anti-H. cyprini serum. Other factors should be examined but we strongly suspect that this newly recognized koi herpesvirus (KHV) has the potential to be a significant cause of mortality among koi and presumably common carp.