W. L. Steffens

Characterization of a new virus from cockatoos with psittacine beak and feather disease

A novel virus isolated from the feather follicles of cockatoos diagnosed as having psittacine beak and feather disease was characterized by electron microscopy, nucleic acid content, and polypeptide composition. Purified virions displayed an icosahedral symmetry, were nonenveloped, and had a mean diameter of 14 to 16 nm negatively stained. Three major viral proteins were identified, with approximate molecular weights of 26.3, 23.7, and 15.9 kDa. The viral nucleic acid was found to be single-stranded DNA based on acridine orange staining, resistance to alkali and ribonuclease, and sensitivity to both DNAse 1 and S1 nuclease. The size of the DNA was estimated to be between 1.7 and 2.0 kb by agarose gel electrophoresis. This size and its circular conformation were confirmed by electron microscopy. A preliminary transmission study using purified virus induced pathological lesions characteristic of those observed in the natural disease. On the basis of the extremely small size of the virions and the single-stranded circular viral DNA, we propose that the etiologic agent of psittacine beak and feather disease represents a previously undescribed viral pathogen.

Comparison of three animal viruses with circular single-stranded DNA genomes.

SummaryNo common antigenic determinants and no DNA sequence homologies were detected when three animal viruses, chicken anaemia agent (CAA), porcine circovirus (PCV), and psittacine beak and feather disease virus (PBFDV), all of which possess circular single-stranded DNA genomes, were compared. Negative contrast electron microscopy showed that PCV and PBFDV particles were 30% smaller than CAA particles and lacked the surface structure of CAA.

ULTRASTRUCTURAL, PROTEIN COMPOSITION, AND ANTIGENIC COMPARISON OF PSITTACINE BEAK AND FEATHER DISEASE VIRUS PURIFIED FROM FOUR GENERA OF PSITTACINE BIRDS

Psittacine beak and feather disease (PBFD) virus, was purified from diseased tissues of a lesser sulphur-crested cockatoo (Cacatua sulphurea), a black palm cockatoo (Probosiger aterrimus), a red-lored Amazon parrot (Amazona autumnalis), and a peach-faced lovebird (Agapornis roseicollis). The histopathology of diseased feathers and follicular epithelium from the different species was compared; basophilic intranuclear inclusion bodies were identified in the follicular epithelium and intracytoplasmic globular inclusions were observed within macrophages located in the feather pulp from the four species. Psittacine beak and feather disease virus antigen was specifically detected by colloidal gold immunoelectron microscopy. The different preparations of purified virions displayed an icosahedral symmetry, were non-enveloped, and had a mean diameter that varied from 12 to 15 nm when negatively stained. Two major viral-associated proteins with approximate molecular weights of 26 and 23 kilodaltons (kd) were consistently demonstrated from the four viral preparations. Purified virions from the four genera were antigenically related. These findings suggest that the PBFD virus purified from numerous genera of diseased birds is similar based on ultrastructural characteristics, protein composition and antigenic reactivity.

Extracutaneous Viral Inclusions in Psittacine Beak and Feather Disease

Thirty-five birds that died with naturally acquired psittacine beak and feather disease (PBFD) were necropsied to identify extracutaneous viral inclusions. Inclusions were found in various tissue sections from 34 of 35 birds. By immunoperoxidase staining, intranuclear and intracytoplasmic inclusion bodies were shown to contain PBFD viral antigen. Inclusion-bearing lesions were widely disseminated but often closely associated with the alimentary tract. Lesions within the palate, esophagus, crop, intestine, bursa of Fabricius, and liver probably serve as sources for viral shedding into the feces.

A Novel DNA Virus Associated with Feather Inclusions in Psittacine Beak and Feather Disease

The nature of feather inclusions was characterized in 32 psittacine birds (30 cockatoos, one peachfaced lovebird (Agapornis roseicollis), and one red-lored Amazon parrot (Amazona autumnalis autumnalis)) with naturally-acquired psittacine beak and feather disease. Intranuclear inclusions within feather epithelial cells and intracytoplasmic inclusions within macrophages in the feather epithelium and pulp cavity contained psittacine beak and feather disease viral antigen when stained by the avidin-biotin complex immunoperoxidase technique. Ultrastructurally, inclusions were observed primarily within macrophages and to a lesser extent within epithelial cell nuclei. Macrophage inclusions appeared as paracrystalline arrays of viral particles. Intranuclear inclusions were less well defined, although scattered viral particles were present. Intracytoplasmic and intranuclear particles in ultrastructural preparations were identified by colloidal gold labeling as psittacine beak and feather disease virus. Feather epithelium was more frequently and severely involved in the disease process than was adjacent follicular epithelium. Plucked feathers with an intact epidermal collar and feather epithelium were preferred to follicular biopsies for histopathologic examination.

Leukocyte changes associated with acute inflammation in chickens.

Leukocyte changes in chickens with turpentine-induced inflammation were investigated sequentially at 0, 6, 12, and 24 hours and at 2, 3, 4, 7, and 14 days. During acute inflammation, significant leukocytosis and heterophilia developed by 6 hours and persisted through 7 days. The peak mean heterophil and leukocyte counts occurred at 12 hours and 3 days, respectively. Left shifts were present at 12 and 24 hours as detected by 100-cell leukocyte differential counts. Heterophil mean nuclear scores documented nuclear hyposegmentation (left shift) during early inflammation and nuclear hypersegmentation (right shift) during convalescence. Mean monocyte and lymphocyte counts peaked at 2 and 3 days, respectively. Basophil and eosinophil counts were erratic. Toxic changes of heterophils were most apparent during intense left shifts and consisted of cell swelling, degranulation, cytoplasmic vacuolation, and cytoplasmic basophilia. Cytoplasmic basophilia was the last aspect of toxic change to resolve. Ultrastructurally, toxic heterophils had intracellular edema, dissolution of granules, retention of ribosomes, nuclear membrane blebs, and decreased heterochromatin density. All inflammation-associated alterations in cell counts and morphology returned to baseline values and appearance by 14 days after turpentine administration.

Detection and confirmation of reptilian adenovirus infection by in situ hybridization

Adenovirus infections are documented in at least 12 different species of reptiles. In contrast to their mammalian and avian counterparts reptilian adenoviruses are not well characterized as to their pathogenic potential and their ability to cause primary disease. In the diagnostic setting, fresh tissues are often not available for virus isolation, and the confirmation of reptilian adenovirus infections is dependent largely upon electron microscopy for the identification of intranuclear viral inclusions associated with histopathologic changes. The diagnosis of adenovirus infection in 2 different species of snake was confirmed by the application of DNA in situ hybridization. Using an aviadenovirus specific oligoprobe, adenoviral DNA was observed in the nuclei of hepatocytes, Kupffer cells, endothelial cells, and enterocytes. Electron microscopy of the liver confirmed the presence of intranuclear viral particles morphologically consistent with an adenovirus. DNA in situ hybridization on formalin-fixed tissues can serve as a suitable alternative to electron microscopy in the diagnosis of reptilian adenovirus infections. Both affected snakes had other concurrent diseases, suggesting that the adenovirus may not have been the primary pathogen.

Infectious anemia caused by a parvovirus-like virus in Georgia broilers.

Pale chicks with necrotic dermatitis, small bursas of Fabricius (BFs), small thymuses, pale bone marrow, and watery blood were suspected of having parvovirus-like virus- (PVLV) associated disease. Histologic lesions included atrophy or hypoplasia of thymuses and BFs, and septic necrotizing clostridial dermatitis and hepatitis. Clostridium perfringens was cultured from skin and liver. A PVLV was isolated in a Mareks disease tumor cell line (MDCC-MSB1) culture and was identified by physicochemical, immunofluorescent, and morphologic features. This isolate was named GA-1 PVLV. Specific-antibody-negative chicks and embryos infected with heat- or chloroform-treated GA-1 PVLV developed anemia at the same rate. Control chicks never were anemic. This is the first isolation of PVLV from clinically ill chickens in the United States and the first report of PVLV-induced anemia in chickens in the Western Hemisphere.

Detection of Eastern Equine Encephalomyelitis Virus RNA in Formalin-Fixed, Paraffin-Embedded Tissues using DNA in Situ Hybridization

Eastern equine encephalomyelitis (EEE) virus was detected in infected formalin-fixed horse and emu tissues and in infected chicken embryo fibroblasts. Results of in situ hybridization using a digoxigenin-labeled 40-base DNA probe complementary to a conserved region of the EEE virus RNA compared favorably with results of both virus isolation and serum neutralization tests. This technique may be useful for diagnosis of EEE virus infection in various animal species, especially when fresh tissues are not available for analysis, and also will provide a means for studying the involvement of alphaviruses in pathogenesis studies.

Polyomavirus Encephalopathy in a Ducorps Cockatoo (Cacatua Ducorpsii) with Psittacine Beak and Feather Disease

Necropsy tissues were examined from an adult wild-caught Ducorps cockatoo (Cacatua ducorpsii) with progressive neurologic signs. Of the tissue specimens selected for histologic evaluation, only the brain contained rare amphophilic, glassy intranuclear inclusions within astrocytes and some neurons. Astrocyte and neuronal degeneration and necrosis also were observed. Scattered astrocytes, with and without discernable inclusions, contained avian polyomavirus (APV) nucleic acid, as determined by DNA in situ hybridization. In addition, endothelial cells and intravascular leukocytes contained psittacine beak and feather disease viral nucleic acid, as determined by DNA in situ hybridization, indicating dual viral infection. Electron microscopic examination of formalin-fixed brain tissue revealed typical intranuclear APV particles in some astrocytes. Encephalopathy ultimately was attributed to APV infection.