Detection and sequence analysis of avian polyomavirus and psittacine beak and feather disease virus from psittacine birds in Taiwan

Avian polyomavirus (APV) and psittacine beak and feather disease virus (PBFDV) are the most common viral diseases of psittacine birds. In Taiwan, however, the existence of these viruses in psittacine birds has not been established. Polymerase chain reaction (PCR) methodology was therefore employed to ascertain whether APV and PBFDV genomes were present in isolates from psittacine birds of Taiwan. A total of 165 psittacine birds belonging to 22 genera were examined between 2002 and 2005. Findings revealed an APV-positive rate of 15.2%, a PBFDV-positive rate of 41.2%, and an APV/PBFDV dual infection rate of 10.3%. After cloning and sequencing, sequences of the PCR products were compared with sequences obtained from GenBank. For APV, the nucleotide identity among VP1 and t/T antigen coding regions ranged from 97.5% to 100% and 97.6% to 100%, respectively. For PBFDV, the nucleotide identity of ORF V1 and ORF C1 sequences ranged from 92.2% to 100% and 83.3% to 100%, respectively. The derived amino acid sequence alignment for PBFDV ORF V1 fragments revealed the conservation of two replication motifs and of the nucleotide binding site motif. In PBFDV, six of 42 deduced positions in the ORF C1 amino acid sequence were considered hypervariable. The established phylogenetic trees based on the four genome fragments examined in this study did not allow the assignment of particular APV or PBFDV nucleotide sequences to distinct avian species.     

Seroprevalence of psittacine beak and feather disease in wild psittacine birds in New South Wales

SUMMARY A haemagglutination inhibition assay was used to detect antibody to psittacine beak and feather disease virus in sera from wild sulphur crested cockatoos (Cacatua galerita), galahs (Eolophus roseicapillus), short-billed corellas (Cacatua sanguinea), eastern long-billed corellas (Cacatua tenuirostris) and other psittacine birds in New South Wales. The seroprevalence of psittacine beak and feather disease ranged from 41% to 94% in different flocks, indicating infection with the virus is widespread in wild populations.     

Psittacine beak and feather disease syndrome in a cockatoo

Psittacine beak and feather disease syndrome was diagnosed in an adult sulfur-crested cockatoo with a history of chronic, progressive feather loss and beak necrosis. A definitive diagnosis was made on the basis of clinical signs and the observation of intracytoplasmic and intranuclear inclusions in involved feather follicular epithelium. Psittacine beak and feather disease syndrome develops in a variety of psittacine species and usually has a progressive and irreversible clinical course. Symmetric feather loss with replacement by severely dystrophic plumage is the salient clinical finding. Beak elongation and breakage also may be found. Treatment of diseased birds remains palliative and consists of a controlled environment, balanced nutrition, antibiotics, and autogenous vaccines. Avian practitioners should include psittacine beak and feather disease syndrome as a potential cause for pathologic feather loss in caged birds.     

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.     

Vaccination and challenge studies with psittacine beak and feather disease virus

SUMMARY: Psittacine beak and feather disease virus (PBFDV) was administered to adult galahs ( Eolophusroselcapillus ) by mouth or by intramuscular injection. Concentration of PBFDV antibodies in serum and excretion of PBFDV were monitored by haemagglutlnation inhibition (HI) and haemagglutination (HA) respectively. After oral administration, 17 of 18 galahs remained clinically normal and a small rise in antibody titre was detected in 3 of 18 birds. After intramuscular administration, antibody was detected in all birds. PBFDV was not detected in the feather dander of birds in either group. One bird developed diarrhoea and high faecal HA titres within 4 days of oral administration and then died. Adult and nestling cockatoos were vaccinated with an experimental inactivated double-oil emulsion vaccine. PBFDV antibody responses are comparable to those induced by a primary-oil emulsion vaccination regimen using Freund's adjuvants. Both vaccines protected nestlings. Three sibling wild-caught sulphur-crested cockatoos were vaccinated but died of PBFD before experimental challenge despite antibody responses in all birds. Unvaccinated control chicks developed acute PBFD within 4 weeks of challenge, probably from PBFDV-induced hepatitis since high concentrations of PBFDV were detected in their livers.     

The pathology of psittacine beak and feather disease

Psittacine beak and feather disease is characterised by loss of feathers, abnormally shaped feathers and overgrowth and irregularity of the surface of the beak. The disease occurs in a number of psittacine species including the Sulphur-crested Cockatoo, Lovebirds , Budgerigars and Galahs . The abnormal appearance of feathers and beak is due to a dystrophic process within the epidermis of the feather and beak. The process consists of epidermal cell necrosis, epidermal hyperplasia and hyperkeratosis. Many of the feather abnormalities are due to retention of a hyperkeratotic feather sheath. A characteristic microscopic finding is the presence of macrophages containing purple intracytoplasmic inclusions in affected epidermis and feather pulp. The inclusions consist of aggregates of particles 17 to 22 nm in diameter. Similar but smaller inclusions occur in epidermal cells. In addition, non-suppurative inflammation occurs in the feather pulp. The findings are suggestive of a viral infection.     

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.     

Psittacine beak and feather disease in three captive sulphur-crested cockatoos (Cacatua galerita) in Thailand

Three sulphur-crested cockatoos (Cacatua galerita) were diagnosed as psittacine beak and feather disease (PBFD). Histopathology of the feather pulp and follicles showed intracytoplasmic botryoid clusters or granular inclusion bodies in epithelial cells and macrophages. Electron microscopy revealed multiple cytoplasmic clusters of electron dense viral particles corresponding to the inclusions. PBFD virus (circovirus) DNA-specific product was detected from formalin-fixed paraffin-embedded feathers by nested polymerase chain reaction (PCR) method.     

Psittacine beak and feather disease virus in budgerigars and ring-neck parakeets in South Africa

Psittacine beak and feather disease (PBFD) is a common disease of the psittacine species and is caused by the psittacine beak and feather disease virus (PBFDV). In this study the occurrence of the disease in ring-neck parakeets and budgerigars in South Africa suffering from feathering problems, using polymerase chain reaction as a diagnostic test was investigated. The genetic variation between viral isolates was also studied. Results indicate that PBFDV can be attributed to being the cause of feathering problems in some of the ring-neck parakeets and budgerigars in South Africa. Genetic variation of isolates occurs between species and individuals. A cheap and easy to use method of blood sample collection on filter paper for diagnostic purposes was also evaluated. It proved to be less stressful to the birds and did not inhibit further processes.     

Routes and prevalence of shedding of psittacine beak and feather disease virus.

Psittacine beak and feather disease (PBFD) virus was recovered from the feces and crop washings from various species of psittacine birds diagnosed with PBFD. High concentrations of the virus also could be demonstrated in feather dust collection from a room where 22 birds with active cases of PBFD were being housed. The virions recovered from the feces, crop, and feather dust were confirmed to be PBFD virus by ultrastructural, physical, or antigenic characteristics. Virus recovered from the feather dust and feces hemagglutinated cockatoo erythrocytes. The specificity of the agglutination was confirmed by hemagglutination inhibition, using rabbit antibodies against PBFD virus. During the test period, 26% (8 of 31) of the birds screened were found to be excreting PBFD virus in their feces, and 21% (3 of 14) of crop washings were positive for PBFD virus. Some birds in the sample group had active cases of diarrhea, whereas others had normal-appearing feces. Diarrhea was found to be the only significant indicator of whether a bird was likely to be excreting virus from the digestive tract. These findings suggest that exposure of susceptible birds to PBFD virus may occur from contact with contaminated feather dust, feces, or crop secretions. Viral particles that were morphologically similar to parvovirus (20- to 24 nm-icosahedral nonenveloped virions) also were recovered from feces of some of the birds.     

A survey to detect subclinical polyomavirus infections of captive psittacine birds in Germany

Infections of avian polyomavirus (APV) are known to cause fatal disease in a wide range of psittacine and non-psittacine birds. Here, we present a survey to investigate the existence of subpopulation of persistent or subclinically infected parrots inside the population of captive psittacine birds in Germany. DNA was isolated from feathers of 85 symptom-free birds from 20 different genera (all psittaciformes) taken from 30 different breeders from all over Germany. The presence of APV was analysed by performing polymerase chain reaction assays (PCR). APV was detected in none of the samples, indicating that the existence of a subpopulation of captive psittacine birds having a persistent APV infection in Germany seems to be relatively low.     

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.     

Cryptosporidiosis in four cockatoos with psittacine beak and feather disease.

Cryptosporidiosis was diagnosed in 4 cockatoos with psittacine beak and feather disease. Three of the birds had cryptosporidiosis confined to the epithelium covering the bursa of Fabricius. One bird had generalized parasitism of the small intestine, large intestine, and bursal epithelium. All of the birds had intermittent to protracted diarrhea before death. Presumably, acquired immunodeficiency from psittacine beak and feather disease promoted establishment of cryptosporidiosis and other secondary diseases including septicemia, peritonitis, chlamydiosis, and mycotic ventriculitis.     

Detection of beak and feather disease virus (BFDV) and avian polyomavirus (APV) DNA in psittacine birds in Italy

Beak and feather disease (psittacine circovirus) and Budgerigar fledgling disease (avian polyomavirus) are viral diseases that can frequently affect captive psittacine birds. We designed the first survey to investigate the presence of beak and feather disease virus (BFDV) and Avian polyomavirus (APV) inside the population of captive psittacine birds in Italy. Samples were collected in 18 Italian psittacine breeding centres and four trade centres over a 4-year period. A total of 1516 birds were tested for BFDV and 877 birds were tested for APV by means of a polymerase-chain-reaction (PCR) assay. BFDV was found in 122 (8.05%) and APV in 7 (0.79%) birds. No significant difference in infection rate was found between imported and locally raised parrots. We report the first BFDV DNA isolation in wild birds imported to Italy from Papua New Guinea.     

A universal polymerase chain reaction for the detection of psittacine beak and feather disease virus.

A universal PCR assay was designed that consistently detected psittacine beak and feather disease virus (BFDV) in psittacine birds affected with psittacine beak and feather disease (PBFD) from different geographic regions across Australia. Primers within open reading frame 1 (ORF1) of the BFDV genome consistently amplified a 717 bp product from blood and/or feathers of 32 birds with PBFD lesions. The PCR did not amplify a product from the feathers or blood from 7 clinically normal psittacine birds. Primers based on regions outside of ORF1 did not consistently produce a PCR product, suggesting there was some genomic variation outside ORF1. The amplified ORF1 PCR products of 10 BFDV isolates, from different psittacine species and from various regions around Australia, were cloned and comparative DNA sequence analysis demonstrated 88-99% of the ORF1 fragments. The derived amino acid sequences of the amplified ORF1 fragments demonstrated similar identity between all 10 isolates. Within ORF1, there was complete conservation of the putative nucleotide binding site and marked conservation of 2 other motifs previously identified as essential components of the replication-associated proteins of other circoviruses and geminiviruses.     

The sensitivities of various erythrocytes in a haemagglutination assay for the detection of psittacine beak and feather disease virus

The erythrocytes of various species were tested in psittacine beak and feather disease (PBFD) virus haemagglutination (HA) and haemagglutination inhibition assays to determine which are suitable for use in these assays. HA activity was observed for erythrocytes of the salmon-crested cockatoo, the sulphur-crested cockatoo, the umbrella cockatoo, the goffin's cockatoo and the cockatiel, with differences amongst individuals within species, but not for erythrocytes of humans, the pig, the guinea pig, the chicken, the goose, the rose-ringed parakeet or the budgerigar. Anti-PBFD virus rabbit sera inhibited the virus-induced agglutination of erythrocytes, confirming the specificity of HA activity. This suggests that selection of suitable psittacine species as well as suitable individuals within a species is necessary when obtaining erythrocytes for the PBFD virus HA assay.     

Laboratory diagnosis of psittacine beak and feather disease by haemagglutination and haemagglutination inhibition

SUMMARY Simple and sensitive haemagglutination and haemagglutination Inhibition assays were developed for psittacine beak and feather disease (PBFD) virus and serum antibody, respectively. The assays were used in the examination of samples from 73 birds clinically affected with PBFD. High antigen titres (log₂ 9 to log₂ 12) were detected In feathers, faeces and cloacal contents of PBFD-affected birds. Antigen was not detected In either faecal or feather samples from 20 normal galahs (Eolophus roselcapillus) and 9 normal sulphur crested cockatoos (Cacatua galerita). After kaolin treatment and haemadsorption of serum, haemagglutination inhibition (HI) antibody titres could not be detected in serum from 42 PBFD-affected birds, whereas serum HI titres from 64 normal psittacine birds ranged from less than log₂ 1 to log₂ 8. Serum and yolk HI antibody responses of 6 PBFD virus-inoculated layer hens were measured. Pre-inoculation chicken sera contained high concentrations of non-specific haemagglutination inhibitors (not detected in chloroform-extracted yolk), which were removed by kaolin treatment and haemadsorption.     

Hemagglutination by psittacine beak and feather disease virus and use of hemagglutination inhibition for detection of antibodies against the virus.

Conditions for psittacine beak and feather disease (PBFD) virus hemagglutination and hemagglutination-inhibition (HI) test reactions are defined. The PBFD virus was found to hemagglutinate cockatoo and some guinea pig erythrocytes. The HI test was used to assay serum antibody titer in birds with active PBFD virus infections and in others that had been exposed to diseased birds. On the basis of HI antibody titers in psittacine birds that had been exposed to PBFD virus, but remained clinically normal, we suggest that some birds exposed to the virus are able to mount an effective immune response. Birds with active PBFD virus infections had lower antibody values than did birds that had been exposed to the virus, but remained clinically normal. On the basis of these findings, the ability to develop a suitable HI antibody response may be crucial in determining the disease status of susceptible birds exposed to the PBFD virus. If HI antibodies are found to have neutralizing activity, then the fact that a high HI titer was induced in birds inoculated with purified PBFD virus might suggest that an immunization program would be effective in preventing PBFD virus infections.