The haemagglutination spectrum of psittacine beak and feather disease virus.

A simple method for concentrating psittacine beak and feather disease virus (PBFDV) from crude feather suspensions is described. The addition of 10% polyethylene glycol (MW 6000 to 9000) to feather suspensions facilitated the precipitation and pelleting of PBFDV by low speed centrifugation. Pellets were resus-pended in one-twentieth of the original volume with caesium chloride (CsCl) buffer and subjected to isopycnic ultracentrifugation. Peak haemagglutination activity (HA) occurred at 1.35 g/ml in PBFDV CsCl gradients. CsCl purified virus agglutinated galah (Eolophus roseicapillus), eastern long-billed corella (Cacatua tenuirostris), sulphur-crested cockatoo (Cacatua galerita), Major Mitchell's cockatoo (Cacatua lead-beateri) and gang gang cockatoo (Callocephalon fimbriatum) erythrocytes, but not those of 19 other avian or five mammalian species. PBFDV agglutinated galah erythrocytes at 4 degrees C and 37 degrees C over a wide range of pH and no change in HA titre was observed when PBFDV was treated with chloroform. HA persisted in PBFDV suspensions heated to 80 degrees C for 30 min, but was not detected after incubation at higher temperatures. High HA titres were detected in the feathers, serum, liver and kidneys of PBFD-affected birds.     

Experimental reproduction of psittacine beak and feather disease/French Moult

Nestling budgerigars and galahs and one-day-old SPF chickens were inoculated with an homogenate prepared from the feathers of a variety of birds with psittacine beak and feather disease (PBFD), and known to contain virus-like particles 20 nm in diameter. Uninoculated birds were included as in-contact controls and groups of birds were also inoculated with homogenate treated with ss-propriolactone to inactivate any virus present. Typical PBFD developed in many of the inoculated birds and in some in-contact controls but in none of the birds inoculated with inactivated homogenate nor in SPF chickens. It is concluded from these findings that PBFD is an infectious disease of viral aetiology and is identical to the disease in budgerigars commonly referred to as 'French Moult'.     

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.     

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.     

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.     

Prevalence and genetic characterization of avian polyomavirus and psittacine beak and feather disease virus isolated from budgerigars in Mainland China

Budgerigar fledgling disease (BFD) and psittacine beak and feather disease (PBFD) are caused by avian polyomavirus (APV) and psittacine beak and feather disease virus (PBFDV), respectively. These diseases frequently infect psittacine birds and result in similar clinical manifestations. In this study, we observed the prevalence of PBFDV infection and a dual infection of APV and PBFDV in a budgerigar (Melopsittacus undulatus) in Mainland China for the first time. One PBFDV isolate and two APV isolates were harvested using chicken embryos. Genetic characterization and phylogenetic analysis of the complete genome of the two APV isolates revealed nucleotide similarity ranging from 99.0% to 99.6% to other sequences in GenBank, and a 14-bp insertion was observed in the genome of one APV isolate. The results of complete genome analysis of the PBFDV isolate showed nucleotide similarity ranging from 83.0% to 95.0% with other PBFDV sequences in GenBank. Genetic characterization and phylogenetic analysis of the APV and PBFDV strains isolated in this study indicated that the isolates from China were closely related to their Japanese counterparts. The results of this study will help to identify molecular determinants and will aid further research on the prevention and control of APV and PBFD infection.     

Reovirus infections associated with high mortality in psittaciformes in The Netherlands.

In The Netherlands between January 2002 and December 2004, numerous psittaciformes died showing severe splenomegaly and hepatomegaly with multifocal acute necrosis. At the start of the outbreaks mostly parakeets were affected, but later larger parrots were also involved. Seventy-eight birds showed the same features and six were examined completely, including a virological examination. Tests for polyomavirus, Pacheco's disease (herpesvirus) and circovirus psittacine beak and feather disease (PBFD) viruses and Chlamydophila psittaci were carried out. All results were negative, except for two cases of circovirus infection. Many concurrent bacterial and parasitic infections were seen. Immunohistochemistry revealed reovirus antigen in intralesional mononuclear cells, and reovirus-like particles could be observed by negative contrast electron microscopy. A reovirus was grown and the isolates reacted with polyclonal reovirus antiserum but did not react with monoclonal antibodies against chicken reovirus. The virus was therefore considered a psittacine reovirus. Because reoviruses were seen consistently, they seemed to be the most probable cause of the outbreaks. Climate, the introduction of new birds and the transportation of birds might be other factors involved in the disease seen in The Netherlands. No regional influence could be seen; therefore, we suggested that the virus might be widespread and carriers could be a source of re-introduction.     

A serologic survey for avian polyomavirus and Pacheco's disease virus in Australian cockatoos.

Sera collected from wild and captive Australian cockatoos and other psittacine species (n = 411) were tested for antibodies to avian polyomavirus (APV) and Pacheco's disease virus (PDV). Of 144 wild sulphur-crested cockatoos (Cacatua galerita) sampled at three regions in New South Wales (NSW) 96 (64.4%) birds had positive (>/= 1:32) neutralizing antibody titres to avian polyomavirus (range 1:32-1:2048). Two of 17 wild long-billed corellas (Cacatua tenuirostris) were also APV-antibody positive. However, no samples from 107 wild galahs (Eolophus roseicapillus) were positive for neutralizing antibody to APV. Sera were also collected from captive psittacine bird flocks from NSW, Tasmania, Victoria, and Western Australia. In a mixed aviary of cockatoos and lorikeets, APV antibody was detected in sera from sulphur-crested cockatoos, Major Mitchell's cockatoos (Cacatua leadbeateri), a white-tailed black cockatoo (Calyptorhynchus baudinii latirostris), a red-tailed black cockatoo (Calyptorhynchus magnificus) a single galah, a rainbow lorikeet (Trichoglossus haematodus), and a scaley-breasted lorikeet (Trichoglossus chlorolepidotus). All 411 wild and captive birds were negative for the presence of neutralizing antibody to PDV. These results indicate that wild sulphur-crested cockatoos in NSW are enzootically infected with avian polyomavirus and that the sampled populations are free of Pacheco's disease.     

Comparitive sensitivity of polymerase chain reaction diagnosis of psittacine beak and feather disease on feather samples, cloacal swabs and blood from budgerigars (Melopsittacus undulates, Shaw 18005).

A longitudinal study was performed in order to investigate virus excretion and viraemia during a clinical outbreak of the psittacine beak and feather disease in budgerigars (Melopsittacus undulatus). Viral nucleic acid was detected in feathers, cloacal swabs and blood samples. Overall, beak and feather disease virus (BFDV) DNA was detected most commonly in feather samples, followed by cloacal swabs, and least frequently from blood samples. In most cases the viraemia was short lived and correlated with clinical signs, such as feather abnormalities. Sequence analysis of the polymerase chain reaction fragment amplified from the replication-associated gene (ORF V1) indicated a close relationship with other BFDV isolates. Overall the highest level of nucleotide identity was found with the ORF V1 of another budgerigar isolate. Our results suggest that feather samples and cloacal swabs should be taken for polymerase chain reaction diagnosis to determine the presence of BFDV in an aviary, but that detection in these samples may not correlate well with psittacine beak and feather disease.     

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.     

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 of novel real-time PCR assays for detecting DNA virus infections in psittaciform birds

Viral diseases of psittacine birds are detected presently by PCR. However, conventional PCR methods are not quantitative and the products can sometimes include non-specific products of the same size. To avoid these problems, real-time PCR assays based on the SYBR Green assay system were developed for the detection and quantitation of four virus diseases of psittacine birds: psittacine beak and feather disease, avian polyomavirus infection, psittacid herpesvirus infection, and psittacine adenovirus infection. Up to 1x10(2) copies of virus DNA were detected, indicating that these assays are as sensitive as conventional PCR assays. The assays are specific because they did not amplify any other pathogens including other viruses, bacteria, and fungi in psittacine birds. The assays measured successfully virus loads in clinical samples (blood, feathers, and tissues), showing that these specimens were suitable targets for the detection and quantitation of viral DNA in psittacine birds.     

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.     

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.     

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.     

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.     

Avian circoviruses of the genus Circovirus: A potential trigger in Pigeon breeder's lung (PBL)/Bird fancier's lung (BFL)

Pigeon breeder's lung (PBL) or Bird fancier's lung (BFL) is one of the most common extrinsic allergic alveolitis or hypersensitivity pneumonitis. It is caused after prolonged inhalation of avian antigens and provokes a hypersensitivity reaction in the lungs of sensitised people. Although the pathogenic mechanism is unclear, the epidemiology of BFL shows that it occurs worldwide, and has been described in adults keeping birds and also in their children. Laboratory findings associated with the disease classified as a type III immunologic reaction that produces blood precipitin antibodies against birds' serum, feathers, intestinal mucin and/or faeces. In particular, the fine dust from pigeon feathers has strong antigenic properties. There is an interaction between host and antigen that seems to be influenced by both genetic and environmental factors. Avian circoviruses (ACV) of the genus Circovirus, has been detected in free-ranging and captive birds worldwide, such as pigeons, canaries, psittacines, Senegal doves, finches, gulls, Australian ravens and geese. T lymphocytes are the main target cells of the ACV and in the above avian species circovirus-like particles were detected in blood, macrophages, feathers, crop secretions, intestinal contents and/or faeces. Most of the ACV was demonstrated that are pantropic and viral antigen in pigeon tissues was most commonly detected in respiratory organs, including the trachea, pharynx and lung. The transmission of the circovirus between the birds usually occurs through inhalation of feathers dust. There is evidence that animal circoviruses may originate when vertebrates become "infected" with DNA from a plant nanovirus. So, it seems that further investigation for the avian circoviruses is needed to determine if they are host specific or not. This study attempts to demonstrate ACV or ACV-like particles as potential triggers in the BFL aetiology, and the possible involvement in BFL's pathogenic mechanism.     

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.     

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.