Beak and feather disease virus infection in cockatiels (Nymphicus hollandicus).

Psittacine beak and feather disease is known to occur in a wide range of psittacine species; however, there are no scientific or credible anecdotal reports of psittacine beak and feather disease occurring in the cockatiel (Nymphicus hollandicus) despite it being one of the world's most commonly kept companion bird species. Consequently, this has resulted in speculation that the species may have some innate resistance to beak and feather disease virus (BFDV) infection. To investigate this hypothesis we conducted a survey of cockatiels (n=88) at commercial aviaries to investigate whether BFDV infection occurs in cockatiels, and found that all birds were virus-free by polymerase chain reaction and haemagglutination assay and had no detectable antibody titre by haemagglutination-inhibition assay. In addition to this, we sequenced the genome of two BFDV isolates obtained from diseased cockatiel feathers and performed cross-reactivity assays using virus eluted from these feathers and sera from naturally immune psittacine birds. Serological cross-reactivity results and phylogenetic analysis of the nucleotide sequences indicated that the cockatiel virus isolates were serologically and genetically different to other BFDV isolates. This is the first paper to report evidence of an antigenically distinct BFDV in psittacine birds.     

Genetic diversity of beak and feather disease virus detected in psittacine species in Australia

The complete nucleotide (nt) sequence of eight isolates of beak and feather disease virus (BFDV) obtained from a range of psittacine species with psittacine beak and feather disease (PBFD) from throughout Australia were compared with the sequences of two BFDV isolates previously reported from Australia (BFDV-AUS) and America (BFDV-USA), respectively. All isolates had the same basic structure including the position of the open reading frames, the hairpin structure between ORF1 and ORF2, the nonanucleotide motif (TAGTATTAC) therein, the three motifs of Rep protein encoded from ORF1 and involved in rolling circle replication, and the P-loop motif previously described, but the genome size of the eight isolates ranged from 1992 to 2018 nt. Overall nt identity of the isolates compared to BFDV-AUS ranged from 84 to 97%; the variation was due to a combination of point mutations and a number of deletions and insertions ranging from 1 to 17 nt in size detected in both coding and noncoding regions. The identity of the nt sequence of ORF2 compared to BFDV-AUS varied from 80 to 99%, while the identity of the deduced amino acid sequences varied from 73 to 99%. Phylogenetic analysis grouped the isolates into four clusters but there were no apparent regional differences or differences related to the psittacine species of origin. While seven ORFs with the potential to encode proteins greater than 8.7 kDa were detected in the BFDV-AUS isolate described previously, only three of these ORFs were detected in all 10 BFDV isolates for which sequence data were available. The three ORFs were ORF1 that presumably encodes the Rep protein, ORF2 presumably the major capsid protein, and the ORF previously designated ORF5. The ORF5 was of two size classes in different isolates, 303 and 474 nt, and only the first 303 nt of the viruses with an ORF of 474 nt were common to the other isolates.     

Nucleotide sequence analysis of a C1 gene fragment of psittacine beak and feather disease virus amplified by real-time polymerase chain reaction indicates a possible existence of genotypes

To investigate sequence diversity of psittacine beak and feather disease virus, samples collected from 31 psittacine species with or without clinical signs were tested for the presence of the viral genome. A real-time polymerase chain reaction was developed amplifying a 202 base pair fragment of the region encoding the capsid protein C1 and detecting 100 to 1000 genome equivalents. The nucleotide sequences of the polymerase chain reaction products showed 84.1 to 100% identity with no consistent pattern with regard to the infected bird species. Amino acid exchanges were concentrated mainly in five of the 42 deduced positions. Sequences obtained from an outbreak of acute beak and feather disease in lories clustered in a separate branch of a phylogenetic tree. Sequences in samples from African grey parrots with feather disorders grouped together, whereas those from the same species with immunosuppression clustered in other branches. These results indicate the possible existence of beak and feather disease virus genotypes.     

Evidence for specificity of psittacine beak and feather disease viruses among avian hosts

Beak and feather disease is a major avian disease of both captive and wild parrot and cockatoo populations. Clinical signs include beak elongation and abnormal growth, together with weight loss and in some individuals the disease is fatal. We investigated the relationship between viral genotypes and their hosts in order to test for a positive association between distinct viral genomes and avian species. Specifically, we used the polymerase chain reaction (PCR) to amplify and sequence a 605-nucleotide (nt) segment of a coding region in the Beak and Feather Disease Virus (BFDV) genome. Feather and blood samples from 25 caged birds representing 10 species were assayed and the BFDV was detected in 21 samples from New Zealand. A phylogenetic analysis of DNA sequences from 17 specimens together with previously published sequences from Australian "isolates" revealed three lineages present in New Zealand. One viral lineage was found in six cockatoos representing two species (designated CT), a second lineage was detected in a budgerigar (designated BG), and a third was found in 10 lorikeets representing seven species (designated LK). This distinctive clustering pattern of viral sequences with groups of psittacine species indicates a genotypic association between the virus and these hosts.     

Comparison of three template preparation methods for routine detection of beak and feather disease virus and avian polyomavirus with single and nested polymerase chain reaction in clinical specimens

Direct comparisons are important when assessing the value of DNA extraction methods for diagnostic virology as the inhibitors present and the efficiency of extraction vary with the target infectious agent as well as the species and the site from which the clinical sample was obtained. Three DNA extraction methods were compared for routine polymerase chain reaction (PCR) detection of beak and feather disease virus (BFDV) in whole blood and feather samples and of avian polyomavirus (APV) in feather samples. Boiling in Chelex® 100 Resin was found to be the most sensitive method for detection of BFDV or APV DNA in both feather and blood samples. In combination with nested PCR it enabled detection of BFDV DNA in 13/13 positive whole blood samples and in 22/23 positive feather samples. It also enabled detection of APV in 31/31 samples detected as positive in this study. NucleoSpin® kits enabled detection of BFDV DNA in only 9/13 blood samples and in 18/23 feather samples. The lower rate of BFDV DNA detection when using NucleoSpin® kits was not a result of inhibition of PCR in most cases. The NucleoSpin® Tissue Kit enabled detection of APV DNA in 29/31 feather samples. Inhibition of DNA amplification was observed when using the DNAzol® Direct kit. Therefore, of the methods evaluated here, Chelex® 100 Resin treatment of samples was the best option for routine testing for BFDV and APV DNA in clinical samples.     

Hematology of vaccinated and non-vaccinated long-billed corellas following infection with beak and feather disease virus (BFDV)

The hematological characteristics of juvenile long-billed corellas (Cacatua tenurostris), with or without prior administration of a psittacine beak and feather disease vaccine, were studied for 97 days after experimental infection with beak and feather disease virus (BFDV). It was found that the pre-challenge hematological values were similar between vaccinated and non-vaccinated corellas. Most pre-challenge parameters were comparable to previously reported values of other cockatoos and psittacine birds. Significant differences were seen in both groups when comparing pre-challenge values with post-challenge values for total and differential leukocyte concentrations, but packed cell volume and total serum protein were not significantly affected by BFDV challenge.     

Development and applications of a monoclonal antibody to a recombinant beak and feather disease virus (BFDV) capsid protein

The development of diagnostic assays for detecting beak and feather disease virus (BFDV) has traditionally been hampered by the difficulty associated with producing suitable reagents, namely purified virus and polyclonal antibodies. In an effort to develop a consistent and standardised source of antibody, a monoclonal antibody to a recombinant BFDV capsid protein has been developed and its use in western blotting, immunohistochemistry (IHC), ELISA and haemagglutination-inhibition (HI) assays characterised. The antibody was specific for both the recombinant BFDV capsid protein and the whole virus and had similar optimal titres when used in western blotting and IHC. The antibody also had HI activity and detected BFDV virus from three genera of psittacine birds, including the recently described cockatiel BFDV isolate. The monoclonal antibody should have widespread application in both research and the development of diagnostic assays for BFDV.     

Identification of a novel circovirus in Australian ravens (Corvus coronoides) with feather disease.

The complete genome of a novel Circovirus isolated from an Australian raven (Corvus coronoides) with feather lesions similar to those that occur in psittacine beak and feather disease is reported. Degenerate polymerase chain reaction primers were designed to amplify and sequence novel Circovirus DNA from affected feathers. Sequence analysis indicated that the tentatively named raven circovirus (RaCV) was 1898 nucleotides in size with two major open reading frames synonymous with other avian circoviruses, ORF C1 and ORF V1, likely to encode a putative capsid protein (Cap) and replicase-associated protein (Rep), respectively. In common with other circoviruses was the conservation of several nucleotide structures and amino acid motifs implicated in virus replication. Comparison with other members of the Circoviridae demonstrated that RaCV shares the greatest sequence homology with canary circovirus (CaCV) and pigeon circovirus (PiCV) and was more distantly related to the beak and feather disease virus, goose circovirus, duck circovirus and the two porcine circoviruses, PCV1 and PCV2. Phylogenetic analysis of the genome and the putative Cap and Rep proteins provided further evidence of the close relationship of RaCV with CaCV and PiCV.     

A unique isolate of beak and feather disease virus isolated from budgerigars (Melopsittacus undulatus) in South Africa

Beak and feather disease virus (BFDV), the causative agent of psittacine beak and feather disease (PBFD) infects psittaciformes worldwide. We provide an annotated sequence record of three full-length unique genomes of BFDV isolates from budgerigars (Melopsittacus undulatus) from a breeding farm in South Africa. The isolates share >99% nucleotide sequence identity with each other and ~96% nucleotide sequence identity to two recent isolates (Melopsittacus undulatus) from Thailand but only between 91.6 and 86.6% identity with all other full-length BFDV sequences. Maximum-likelihood analysis and recombination analysis suggest that the South African budgerigar BFDV isolates are unique to budgerigars, are non-recombinant in origin, and represent a new genotype of BFDV.     

Molecular analysis and associated pathology of beak and feather disease virus isolated in Italy from young Congo African grey parrots (Psittacus erithacus) with an “atypical peracute form” of the disease

This study is the first report on the genetic and pathogenic characterization of beak and feather disease virus (BFDV) occurring in Italy. Twenty BFDV strains isolated in Italy from juvenile Congo African grey parrots (Psittacus erithacus) were investigated. Seventeen strains showed an “atypical peracute form” (aPF) of the disease, and three a chronic form (CF). The birds with aPF had been weaned, were independent as far as food and protection were concerned and apparently were without lesions. The gene coding for the putative coat protein was amplified in all isolates while the BFDV genome was sequenced completely in 10 samples, eight of them belonging to aPF affected birds and two from CF of the disease. All full genomes clustered into the J strain of BFDV, where two new subtypes were identified. Recombination analyses showed evidence of genetic exchanges in two BFDV genomes. In addition, a correlation between viral isolate and origin of the breeding material was shown, while an association between the genetic features of the virus and the clinical form was not observed. Histologically, apoptosis was detected frequently in aPF samples and sporadically in CF samples. Interestingly, BFDV antigens were detected in the nuclei and cytoplasm of such apoptotic cells. The data presented here support the hypothesis that, in the absence of a defined BFDV genetic variant accountable for a specific clinical form of psittacine beak and feather disease, differences in the apoptotic rate between aPF and CF are strictly host related.     

Baculovirus expression of beak and feather disease virus (BFDV) capsid protein capable of self-assembly and haemagglutination

Beak and feather disease virus (BFDV) is a common avian circovirus infection of wild Psittaciformes and is a recognised threat to endangered psittacine species. Currently, there is a requirement to develop BFDV antigen for diagnostic purposes and since efforts to propagate BFDV in vitro have so far been unsuccessful the entire coding region of BFDV ORF C1 was expressed in Sf9 insect cells using a baculovirus expression system. The entire coding region of BFDV ORF C1, the presumptive capsid, was expressed in Sf9 insect cells using baculovirus expression system. Electron microscopic examination of negatively stained material demonstrated that the recombinant protein self-assembled to produce virus-like particles (VLPs) thus confirming that ORF C1 is likely to be the sole determinant for capsid construction in vivo. BFDV VLPs also possessed haemagglutinating activity which provides further evidence that self-assembled BFDV VLPs retain receptor mediated biological activity and that the determinants for BFDV haemagglutination activity rely solely on the capsid protein. The recombinant protein reacted with anti-BFDV sera from naturally immune parrots and cockatoo and from chickens experimentally inoculated with native BFDV in both Western blots and haemagglutination inhibition (HI) assay. BFDV VLPs were also a suitable replacement antigen for serological detection of BFDV antibody by HI.     

Recombinant expression of a truncated capsid protein of beak and feather disease virus and its application in serological tests.

Beak and feather disease virus (BFDV) causes severe disease characterized by irreversible feather disorders and severe immunosuppression in many psittacine species. BFDV cannot be propagated in tissue or cell cultures, rendering virus propagation and thus diagnosis rather difficult. To develop reliable diagnostic methods, the region encoding the BFDV capsid protein C1 was cloned from an infected sulphur-crested cockatoo (Cacatua galerita). Phylogenetic analysis showed this gene had 76.3 to 83.2% amino acid identity to published sequences. No protein was detected after induction of full-length C1 expression in Escherichia coli. However, deletion of an amino-terminal arginine-rich sequence facilitated expression. C1(39-244)-His, a polyhistidine-tailed variant of this protein, was purified and used for immunization of chickens. The immune sera detected C1 with an apparent molecular weight of 27 kDa in western blots of organ homogenates of BFDV-infected birds. Using C1(39-244)-His as antigen, 11 psittacine sera were tested for the presence of BFDV-specific antibodies by enzyme-linked immunosorbent assay and immunoblotting. The results obtained correlated well with the BFDV-specific haemagglutination inhibition activity of the sera, suggesting C1(39-244)-His has value as a recombinant antigen for BFDV-specific serological tests.     

Genetic diversity of the Rep gene of beak and feather disease virus in South Africa

Summary. A study on the genetic variation of Beak and feather disease virus (BFDV) isolates in South Africa was performed by amplifying and sequencing a region within the ORF1 of the genome. Six different BFDV isolates were found in 15 psittacine species from 6 regions within South Africa, representing three unique isolates and three isolates that clustered into a budgerigar lineage (BG) previously described.     

Genome sequence determinations and analyses of novel circoviruses from goose and pigeon

The genomes of novel circoviruses from goose and pigeon, which were isolated using degenerate primer and inverse primer PCR methods, were cloned and sequenced. Comparative nucleotide (nt) sequence analyses showed that the goose circovirus (GCV) and pigeon circovirus (PiCV) possessed genomes which were 1821 and 2037 or 2036 nt, respectively, and which had features in common with the genomes of porcine circoviruses types 1 and 2 (PCV1, PCV2) and psittacine beak and feather disease virus (BFDV), such that they can now be assigned to the genus Circovirus of the family Circoviridae. Common features include the possession of (i) a potential stem-loop/nonanucleotide motif with which the initiation of rolling circle replication of the virus DNA is associated; (ii) two major ORFs, located on the virus (V1 ORF) and complementary (C1 ORF) strands, which encode the replication-associated protein (Rep) and capsid protein, respectively; (iii) high levels of amino acid identity (41.2--58.2%) shared with other circovirus Rep proteins; and (iv) direct/inverted repeat sequences within the putative intergenic region. On the basis of nt and amino acid sequence identities, GCV is substantially less closely related to BFDV than PiCV is to BFDV.     

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.     

A feather disease in Senegal doves (Streptopelia senegalensis) morphologically similar to psittacine beak and feather disease.

The pathogenesis and epidemiology of a feather disease in wild Senegal doves (Streptopelia senegalensis) which is morphologically similar to psittacine beak and feather disease (PBFD) was investigated. Although the lesions in doves resembled PBFD there was little evidence for the presence of psittacine circovirus (PsCV). Haemagglutination activity (HA) using type A galah (Eolophus roseicapillus) erythro-cytes was not detected in feathers or livers of affected doves as would occur in PBFD. Low concentrations of HA excreted in the faeces of affected doves was not caused by psittacine circovirus (PsCV) because the antigen in faeces also caused haemagglutination of PsCV-insensitive type B galah erythrocyte and was not inhibited by anti-PsCV antibody. Similar HA of unknown cause was also detected in faeces from clinically normal Senegal doves. Anti-PsCV haemagglutination inhibiting (HI) antibody was not detected in the serum of affected doves or in the blood of 206 clinically normal wild Senegal doves or 17 captive columbid birds in close contact with a flock of psittacine birds that was known to be PsCV-infected. Senegal doves also failed to seroconvert after two inoculations with PsCV purified from the feathers of a PBFD-affected long-billed corella (Cacatua tenuirostris). The results indicate that the feather disease seen in feral Senegal doves in Perth is not due to PsCV although the possibility that it is due to another antigenically distinct circovirus was not eliminated.     

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.     

Psittacine beak and feather disease infected cells show a pattern of apoptosis in psittacine skin.

Skin biopsies from 23 birds with psittacine beak and feather disease (PBFD) were examined by light and electron microscopy. Affected cells, preferentially found in the cell layers of the feather follicles, could be clearly identified by the presence of intracytoplasmic virus inclusion bodies. Ultrastructurally, the degenerative process in these cells was morphologically suggestive of apoptosis.     

Nucleotide sequence-based identification of a novel circovirus of canaries

Circovirus-like, spherical particles measuring 16 to 18 nm in diameter were detected in organ homogenates from adult canaries that had died after a short illness characterized by dullness, anorexia, lethargy and feather disorder. A polymerase chain reaction method, based on degenerate primers specific to conserved amino acid sequences in the circovirus replication-associated protein, was used to amplify DNA specific to a novel circovirus, tentatively named canary circovirus (CCV). Sequence analysis of a 510 nucleotide genomic fragment indicated that CCV exhibited 67.4, 64.9, 53.7 and 53.9% nucleotide identities and 70.0, 61.8, 40.4 and 40.1% amino acid identities with columbid (pigeon) circovirus (CoCV), beak and feather disease virus (BFDV), porcine circovirus type 1 and porcine circovirus type 2, respectively. CCV therefore represents a new avian virus of the genus Circovirus of the family Circoviridae, and is more closely related to CoCV than BFDV.The availability of nucleotide sequence data will facilitate the development of DNA-detecting diagnostics with which the prevalence of CCV infections can be assessed.