Histopathologic Changes, Ultrastructure, and Molecular Characterization of an Adenovirus in a Sun Conure (Aratinga solstitialis).

In this case report, we describe the pathologic changes and the ultrastructural and molecular characteristics of an adenovirus in a sun conure (Aratinga solstitialis) that presented with a history of sudden death. On histologic examination, there was multifocal hepatic and splenic necrosis. Within some hepatocytes and unidentified cells in the spleen, renal interstitial fibroblasts, and ovarian stroma were intranuclear amphophilic inclusion bodies. Electron microscopy of affected tissue showed intranuclear icosahedral viral particles with an inner capsid (29.2-33.8 nm in diameter) and an outer capsid (70.2-71.7 nm in diameter). Next-generation sequencing and BLAST analysis of complementary DNA synthesized from RNA extracted from formalin-fixed tissues showed an adenovirus, designated sun conure adenovirus (SCAdv). A DNA in situ hybridization (ISH) probe, constructed from the SCAdv and similar sequences from GenBank, was also positive in the intranuclear inclusion bodies, whereas standard ISH for psittacine adenovirus 1 was negative. These results show that ancillary diagnostic testing, such as next-generation sequencing, even using formalin-fixed, paraffin-embedded tissues, along with ISH, can be useful in identifying additional, unknown viruses that show similar pathology to commonly known viruses but do not show up as positive on routine diagnostic tests.

Reporte de caso- Cambios histopatológicos, ultraestructura y caracterización molecular de un adenovirus en una cotorra solar (Aratinga solstitialis). En este reporte de caso, se describen los cambios patológicos y las características ultraestructurales y moleculares de un adenovirus en una cotorra solar (Aratinga solstitialis) que se presentó con un historial de muerte súbita. En el examen histológico, hubo necrosis hepática y esplénica multifocal. Dentro de algunos hepatocitos y células no identificadas en el bazo, los fibroblastos intersticiales renales y en el estroma ovárico se encontraron cuerpos de inclusión anfofílicos intranucleares. La microscopía electrónica del tejido afectado mostró partículas víricas intranucleares icosaédricas con una cápside interna (de 29.2 a 33.8 nm de diámetro) y una cápside externa (de 70.2 a 71.7 nm de diámetro). Mediante el análisis de secuenciación de segunda generación y por la Herramienta de Búsqueda de Alineaciones Local Básica (con siglas en inglés BLAST) del ADN complementario sintetizado a partir de ARN extraído de tejidos fijados con formalina mostraron un adenovirus, denominado adenovirus de cotorra solar (SCAdv). Se construyó una sonda de ADN para hibridación in situ (ISH), a partir de la secuencia del virus SCAdv y de secuencias similares de GenBank, que generó reacción positiva en los cuerpos de inclusión intranucleares, mientras que la hibridación in situ estándar para el adenovirus I de psitácidos fue negativa. Estos resultados muestran que las pruebas de diagnóstico complementarias, como la secuenciación de segunda generación, utilizando tejidos fijados con formalina e incluidos en parafina junto con la hibridación in situ pueden ser útiles para identificar virus adicionales desconocidos que muestran una patología similar a los virus comúnmente conocidos, pero que no se detectan con las pruebas diagnósticas de rutina.

Proliferative pododermatitis associated with virus-like particles in a northern gannet.

Small multifocal lesions of proliferative pododermatitis were observed in an emaciated adult male northern gannet (Morus bassanus). Ultrastructurally, these lesions were associated with numerous virus-like particles with a size and morphology suggestive of Papovaviridae. DNA in situ hybridization with probes for avian polyomaviral and papillomaviral nucleic acid and an immunohistochemical test for the presence of papillomaviral antigen failed to identify this virus further. To our knowledge, papovavirus-like particles have not been recognized previously in this avian species.

Use of an inactivated virus vaccine to control polyomavirus outbreaks in nine flocks of psittacine birds.

Nine flocks of psittacine birds were examined because of sudden death of neonates. In each flock, cause of death was determined to be polyomavirus infection, by means of DNA testing and in situ hybridization. Contaminated areas of aviaries were cleaned and disinfected, and vaccination programs, using a recently approved inactivated polyomavirus vaccine, were instituted. Use of the vaccine was found to be safe and efficacious.

Safety, immunogenicity, and efficacy of an inactivated avian polyomavirus vaccine.

OBJECTIVE: To determine safety, immunogenicity, and efficacy of an inactivated avian polyomavirus vaccine in nonbudgerigar psittacine birds that varied in age, species, and immunologic status. ANIMALS: Safety of the vaccine was evaluated in 1,823 psittacines representing more than 80 species. Immunogenicity was evaluated in 285 birds (260 of various Psittaciformes species, 25 chickens). Efficacy was evaluated in 104 birds (78 of various Psittaciformes species, 26 chickens). PROCEDURES: Safety was evaluated by vaccinating birds that were determined to be seronegative or seropositive (titer > 1:10) prior to vaccination. Birds were then evaluated for clinically detectable systemic or local reactions for 2 months to 2 years. Immunogenicity was evaluated by testing for virus-neutralizing antibodies, vaccinating each bird twice, and then testing for a significant change in antibody titer. Efficacy was evaluated by vaccinating birds, followed in 2 to 4 weeks by intramuscular or intravenous challenge exposure. After challenge exposure, protection was evaluated by attempting to recover virus from tissues or by observing birds for clinical signs of disease and testing for a significant change in titer. CONCLUSIONS: Avian polyomavirus vaccine is safe, immunogenic, and efficacious for use in multiple species of mature and immature psittacines. CLINICAL RELEVANCE: Until now, prevention of polyomavirus infection in psittacine birds could only be accomplished through strict isolation to reduce potential exposure to the virus. The USDA-registered inactivated avian polyomavirus vaccine can safely be used to protect vaccinates from infection and control spread of this virus in flocks.

Diagnosis of avian adenovirus infections using DNA in situ hybridization.

Three DNA oligonucleotide probes designated FN-23, FN-48, and FN-96 were evaluated for the diagnosis of aviadenovirus infections by DNA in situ hybridization. Paraffin-embedded tissues were obtained from birds with confirmed adenovirus infection, birds with putative adenovirus infections, and birds with intranuclear inclusions caused by herpesvirus and polyomavirus. In birds with confirmed adenovirus infection, probes FN-23 and FN-96 identified 78% and 72% of diseased individuals, respectively. Only probe FN-48 detected chickens with group II adenovirus infection. In birds with putative adenovirus infection, the DNA probes confirmed aviadenovirus infections in 76% of the population. Probes FN-23, FN-96, and FN-48 detected 85%, 74%, and 18% of adenovirus-infected birds, respectively. None of the DNA probes cross-hybridized with tissues from polyomavirus-infected psittaciform birds or with tissues from a chicken with infectious laryngotracheitis. In contrast, probe FN-23 did cross-hybridize to herpesvirus-infected tissues from two of eight psittaciform birds with Pacheco's parrot disease. Probes FN-48 and FN-96 did not react with these tissues.

An inactivated avian polyomavirus vaccine is safe and immunogenic in various Psittaciformes.

The safety and immunogenicity of adjuvanted and nonadjuvanted inactivated avian polyomavirus vaccines, administered either intramuscularly or subcutaneously (s.c.), were evaluated in a group of mixed species Psittaciformes. In 233 vaccinates representing species of macaws, cockatoos, conures, and parrots, gross reactions were limited to small scab formation at the s.c. injection site in three African grey parrots. Both vaccines stimulated a virus neutralizing (VN) antibody response, particularly in birds that were seronegative prior to vaccination. Ninety-three percent of the birds that were seronegative at the beginning of the study seroconverted (greater than fourfold increase in VN antibody titer) by 2 weeks after the second vaccination. Seventy-six percent of all the vaccinates had at least a fourfold increase in VN antibody titer at this time. There was no significant difference in seroconversion between the birds vaccinated with adjuvanted or nonadjuvanted vaccines. This study indicates that an inactivated avian polyomavirus vaccine can be used to safely immunize various species of psittacine birds in a field setting.

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.

Histologic evaluation of the crop for diagnosis of proventricular dilatation syndrome in psittacine birds.

Histologic sections of crop tissue were evaluated for the presence of lymphoplasmacytic infiltrates within mesenteric ganglia. All birds with proventricular dilatation syndrome that had lymphoplasmacytic infiltrates in crop ganglia had similar infiltrates in the proventricular and/or ventricular ganglia. False-negative crop biopsy results occurred approximately 24% of the time. More invasive procedures, such as proventricular or ventricular biopsy, may be necessary if the crop biopsy is nondiagnostic in a bird with clinical signs of proventricular dilatation syndrome.

Diagnosis of psittacine beak and feather disease (PBFD) viral infection, avian polyomavirus infection, adenovirus infection and herpesvirus infection in psittacine tissues using DNA in situ hybridization.

The evaluation of the usefulness of DNA probes in a diagnostic setting to identify nuclear inclusions in selected viral infections (psittacine beak and feather disease viral infection, avian polyomavirus infection, adenovirus infection and Pacheco's parrot disease) is reported. A DNA in situ hybridization method was used to detect viral nucleic acid in sections of paraffin-embedded tissues coming from birds naturally and/or experimentally infected. It is concluded that DNA probes used for polyomavirus (FN-19) and adenovirus (FN-23) are able to identify nucleic acid of each virus in the cells with nuclear inclusions, and when used for psittacine beak and feather disease virus (FN-8), and Pacheco's parrot disease virus (FN-49) are able to detect viral nucleic acid in cells with or without inclusions.

Diagnosis of polyomavirus infection in seedcrackers (Pyrenestes sp.) and blue bills (Spermophaga haematina) using DNA in situ hybridization.

Avian polyomavirus (APV) infection was diagnosed in a closed research colony of seedcrackers (Pyrenestes sp.) and blue-bills (Spermophaga haematina) using DNA in situ hybridization. The DNA probe was a 1-kbp double-stranded PCR-generated probe that recognized a conserved nucleotide sequence within the VP-1 gene. Using this technique, APV infection was diagnosed, in 25 of 45 birds based upon examination of formalin-fixed paraffin embedded tissues. Birds infected with APV apparently had a higher incidence of hepatic necrosis, hepatitis, bacterial infections and parasitism than did birds without APV.

Diagnosis of polyomavirus-induced hepatic necrosis in psittacine birds using DNA probes.

Liver sections from 32 psittacine birds with multifocal to coalescing hepatocellular necrosis were examined to determine the cause of disease. Avian polyomavirus (APV) infection (19 of 32 birds), bacterial hepatitis (5 of 32 birds), and chlamydiosis (3 of 32 birds) were major causes of hepatic disease. The presence of APV inclusions or nucleic acid was demonstrated using hematoxylin and eosin (HE) staining, DNA in situ hybridization, and DNA amplification with Southern blotting. Amphophilic intranuclear inclusions, suggestive of APV infection, were observed in HE-stained liver sections from 5 of 32 birds. Hepatocellular karyomegaly was present in liver tissues from 10 birds (5 birds with typical APV inclusions and 5 birds without discernable inclusions). DNA in situ hybridization recognized intranuclear APV nucleic acid in liver sections of 18 of 32 birds. DNA amplification with Southern or dot blots also identified APV nucleic acid in processed, paraffin-embedded livers of 18 of 32 birds. This study demonstrates that acute APV infection is a frequent cause of multifocal to coalescing hepatocellular necrosis in psittacine birds. Furthermore, APV infection is best diagnosed using DNA probes, especially when typical intranuclear inclusions are not observed microscopically.

Production and characterization of monoclonal antibodies to psittacine beak and feather disease virus.

Monoclonal antibodies specific for the virus that causes psittacine beak and feather disease (PBFD) were produced by fusing spleen cells from mice immunized with purified concentrated PBFD virus with mouse myeloma cell line Sp2/0. The resulting hybridomas were tested for reactivity against whole purified virus by an enzyme-linked immunosorbent assay (ELISA) system. Four clones, designated 15H8, 8E3, 11G12, and 2C3, were subcloned by limiting dilution. Isotyping indicated that clone 15H8 was secreting IgG, whereas the remaining clones secreted IgM. The secreted immunoglobulins were characterized by reactivity against purified PBFD virus using immunoblotting procedures, by immunohistochemical staining of virus-induced lesions in infected tissues, and by inhibition of PBFD virus agglutination of cockatoo erythrocytes. Antibodies secreted by clones 15H8 and 8E3 had the strongest activity against purified whole virus. Only immunoglobulin secreted by the clone 15H8 could be used to detect viral antigen in infected tissues. None of the monoclonal antibodies had hemagglutination-inhibition activity.

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 peach-faced 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.

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.

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.

Myocardial sarcocystosis in a grand eclectus parrot (Eclectus roratus) and a Moluccan cockatoo (Cacatua moluccensis).

Cardiac sarcocystosis is described in a grand eclectus parrot and a Moluccan cockatoo. Many cysts containing metrocytes were observed within cardiac muscle fibers on tissue sections stained with hematoxylin and eosin. Characteristic ultrastructural features of the cyst walls included the presence of villous projections containing microtubules. Compartmentalization of the cysts resulted from inward extensions of the cyst wall. The differential diagnosis of sarcocystosis, the life cycle of the parasite, and control measures are discussed.

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.