A comparison of haemagglutination, haemagglutination inhibition and PCR for the detection of psittacine beak and feather disease virus infection and a comparison of isolates obtained from loriids

Bahman Khalesi, Nicolai Bonne, Meredith Stewart, Margaret Sharp and Shane Raidal

Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150, Australia

Correspondence
Shane Raidal
raidal{at}murdoch.edu.au


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Psittacine beak and feather disease (PBFD) is recognized as a threat for endangered psittacine birds in Australia, New Zealand and South Africa. Several diagnostic methods for the detection of beak and feather disease virus (BFDV) infection have been developed but there are few studies comparing the relative merits or sensitivity and specificity of each diagnostic test. In this report, the results of PCR, haemagglutination (HA) and haemagglutination inhibition (HI) testing of diagnostic samples collected from 679 samples from a range of psittacine bird species suspected of being infected with BFDV are summarized and compared. There was a strong agreement (kappa = 0·757; P<0·0001) between PCR and HA testing of feather samples and PCR-negative birds were 12·7 times more likely to have HI antibody than PCR-positive birds. False-positive HA results with titres up to 1 : 320 were identified in six feather samples that were PCR negative; the haemagglutination detected in these samples was not inhibited by anti-BFDV antisera and was removed by filtration through a 0·22 µm filter. Similarly, one false-negative PCR result was detected in a feather sample that had a high HA titre (>1 : 40 960) and four false-positive PCR results were detected in a batch of four feather samples. Of 143 birds that were feather PCR positive, only two had detectable HI antibody, and these birds were also feather HA negative, suggesting that they were developing immunity to recent infection. All birds with HI antibody were negative on feather HA testing. The assays confirmed BFDV infection in two endangered swift parrots (Lathamus discolor) and phylogenetic analysis of the sequence data generated from ORF V1 of these isolates provide further evidence of BFDV genotypes clustering in parallel with the Loriidae, Cacatuidae and Psittacidae.

The GenBank/EMBL/DDBJ accession numbers for the novel BFDV ORF V1 sequences determined in this study are DQ016388–DQ016396.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Psittacine beak and feather disease (PBFD) is the most common viral disease of wild psittaciformes (cockatoos, lorikeets and parrots) in Australasia and it has a worldwide distribution in captive birds. It is recognized as a threat for endangered psittacine birds in Australia, New Zealand and South Africa. In Africa, PBFD has been identified in captive Agapornis spp. (Kock, 1990) as well as wild cape parrots, Poicephalus robustus (Heath et al., 2004), although these outbreaks may have been caused by the release of aviary birds. The disease is also widespread in captive collections of psittacine species in Europe and the USA.

The causative agent, Beak and feather disease virus (BFDV), has a circular ssDNA ambisense genome (Bassami et al., 1998; Niagro et al., 1998) with two major ORFs. ORF V1 encodes the replication-associated protein (Rep) and ORF C1 encodes the capsid protein. BFDV is a haemagglutinating circovirus, which has permitted the development of haemagglutination (HA) and haemagglutination inhibition (HI) assays for the virus and antibody responses to infection, respectively (Raidal & Cross, 1994a). These assays, as well as PCR testing based on the relatively conserved ORF V1 (Ypelaar et al., 1999; Ritchie et al., 2003), are in wide use throughout Australia and elsewhere in the world for diagnosing infection (Raidal et al., 1993a; Sanada & Sanada, 2000), but there have been no studies comparing the value of HA and HI testing relative to PCR. We decided to compare the three tests on feather and blood samples sent to us for routine diagnostic testing. We also sequenced PCR products from isolates obtained from loriids to investigate further the current debate over the emergence of genetically adapted strains in lorikeets, parrots and cockatoos (Ritchie et al., 2003; Raue et al., 2004; de Kloet & de Kloet, 2004; Heath et al., 2004).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Samples.
Feather and blood samples were submitted by referring veterinarians throughout Australia from 623 psittacine birds tentatively diagnosed with PBFD or with a history of being in contact with PBFD-affected birds. Some samples were submitted to confirm absence of disease in quarantined birds. Blood samples were either collected onto filter paper as described by Riddoch et al. (1996) when long-distance transportation was required or collected into heparinized collection tubes for processing in our laboratory (Raidal et al., 1993a). Developing feathers or dried feathers with lesions were collected and transported unpreserved.

To compare feather and blood PCR testing, a flock of 56 peach-faced lovebirds recently imported into a pet shop was sampled by feather HA, feather PCR, blood PCR and HI antibody testing as described below.

HA and HI assays.
HA assays were performed on feather samples using galah (Eolophus roseicapillus) erythrocytes and BFDV antigen derived from a sulphur-crested cockatoo (Cacatua galerita) as described by Raidal et al. (1993a). HI assays were performed on blood samples collected onto filter paper as described by Riddoch et al. (1996) or as described by Raidal et al. (1993a) when submitted as plasma or serum. Confirmation of HA results by inhibition of HA activity with BFDV-specific antibody (Raidal et al., 1993a), filtration of samples through 0·22 µm filters and/or parallel testing with BFDV-insensitive galah erythrocytes was carried out as necessary whenever there was a discrepancy between HA and PCR test results. A comparative analysis of the data was performed using chi-squared tests for proportions using SPSS 4.0.

In an attempt to identify serotypes of BFDV, HA cross-reactivity was assessed by performing HI assays on eight different BFDV isolates obtained from two rainbow lorikeets (Trichoglossus haematodus), a musk lorikeet (Glossopsitta concinna), a red lory (Eos bornea), two swift parrots (Lathamus discolor), a sulphur-crested cockatoo and a scarlet-chested parrot (Neophema splendida) with blood samples containing known HI antibody titres (> 320 HIU per 50 µl) obtained from seven different psittacines [two short-billed corellas (Cacatua sanguinea), a sulphur-crested cockatoo, two rainbow lorikeets, one red lory and one galah–corella hybrid].

Preparation and purification of DNA from feather and blood samples.
DNA was extracted from feather tissues using modified methods of Taberlet & Bouvet (1991) and Morin et al. (1994) as described previously by Ypelaar et al. (1999). DNA was extracted from the blood using the QIAamp DNA blood mini kit (Qiagen).

Limit of detection of BFDV by HA and PCR assays.
To determine the limit of detection of the HA and PCR assays, serial 1 : 10 dilutions were prepared of an initial 10 % (w/v) suspension made from diseased feathers obtained from a long-billed corella (Cacatua pastinator) with chronic PBFD and each dilution was tested as described above for HA and below (Ypelaar et al., 1999) for PCR.

Amplification and analysis of BFDV.
The PCR assay was performed as described by Ypelaar et al. (1999) with forward primer 5'-AACCCTACAGACGGCGAG-3 and reverse primer 5'-GTCACAGTCCTCCTTGTACC-3, used to amplify a segment of BFDV ORF V1. All PCR products generated were visualized by agarose gel electrophoresis and a positive result was determined visually. Selected PCR amplicons of interest for DNA sequencing were purified from the agarose using the QIAquick gel extraction kit (Qiagen) and were ligated into pCR2.1 vector (Invitrogen) according to the manufacturer's protocol. The ABI Prism Dye Terminator cycle sequencing kit (Applied Biosystems) was used according to the manufacturer's protocols except that reaction volumes were halved, to 10 µl, and the annealing temperature was raised to 58 °C. Sequence information was determined using an Applied Biosystems 3730 DNA Analyser.

DNA sequencing of BFDV isolates from swift parrots and lorikeets.
The generated BFDV ORF V1 sequences were edited and assembled using SeqEd version 1.0.3 (Applied Biosystems) with corrections made on base-pair differences based on the chromatograms. All sequences were analysed using a range of programs provided by the Australian National Genomic Information Service (ANGIS) and the National Center for Biotechnology Information (NCBI). The details of the new isolates and reference isolates with their GenBank accession numbers are summarized in Tables 1 and 2.


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Table 1. GenBank accession numbers for ORF V1 DNA sequences generated from 9 BFDV-infected lorikeets and swift parrots

Feather HA (HAU per 50 µl) and blood HI titres (HIU per 50 µl) are shown.

 

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Table 2. Circovirus reference sequences from psittacine and non-psittacine hosts used in this study

 
Edited sequences were analysed using the BLASTN and BLASTP programs (Altschul et al., 1997) at the NCBI (http://www.ncbi.nlm.nih.gov/blast/) and compared with other previously published BFDV ORF V1 sequences. The circovirus ORF V1 nucleotide and amino acid sequences were aligned using the program CLUSTAL X 1.8 (Thompson et al., 1997). Phylogenetic analyses were preformed using the neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) procedures with PAUP 4.0b2 and the Tajima–Nei model for distance estimation. A phylogenetic tree rooted to canary circovirus (Phenix et al., 2001) was constructed using the TreeCon 1.3b program with 1000 bootstrap cycles (Felsenstein, 1985).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
HA and PCR assays limit of detection
HA titres obtained from serial tenfold dilutions of feathers obtained from a corella with PBFD were >40 960, 25 600, 32 000, 40 000, <200 000 and <2 000 000 HAU per 50 µl. A positive PCR result was obtained for the 1 : 10, 1 : 100 and 1 : 1000 but not in the 1 : 100 000 or 1 : 1 000 000 dilutions on the same material.

Comparison of HA, HI and feather PCR assay for the detection of BFDV
A total of 623 diagnostic accessions were received, but not all accessions provided appropriate samples for all three tests. Of 621 feather samples received, 143 (23 %) were PCR positive (Table 3). There was a strong agreement between the PCR and HA tests (kappa = 0·757; P<0·0001) and, of the 143 feather samples, 132 were also tested by HA and 88 (66·7 %) were also positive, with HA titres ranging from 1 : 80 to 1 : 40 960 (mean log2 10·4 ± 2·6 HAU per 50 µl) and 44 were HA negative but PCR positive. Of the remaining feather samples that were PCR negative, six were initially positive by HA, with titres up to 1 : 320, but the false HA in these samples was not inhibited by anti-BFDV antisera and was removed by filtration through a 0·22 µm filter, which indicated that BFDV was not the cause of the HA in the sample. Suspected false-positive PCR results were obtained on a batch of feather samples from four clinically normal birds that were subsequently PCR negative on repeat retesting (Table 3), and a first-round false-negative PCR result was detected in one bird that had a clinical description of PBFD and a very high feather HA titre (Table 3). Table 4 provides the prevalences of BFDV feather excretion according to the major psittaciform groups.


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Table 3. Test results obtained from diagnostic samples submitted from 623 psittacine birds showing the numbers of birds in each group

False-positive and false-negative results are italicized.

 

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Table 4. Prevalence of BFDV shedding in feathers based on HA and or PCR testing of samples collected from 623 birds of various families and genera

 
There was a poor agreement between the PCR and HI test results (kappa = –0·193; P<0·0001), with PCR-negative birds 12·7-fold (95 % CI 3·1–52·6) more likely to be HI positive than PCR-positive birds. Of the 143 birds that were feather PCR positive, only two had detectable HI antibody and these birds were also feather HA negative, suggesting that they were developing immunity to recent infection. Otherwise, HI antibody was detected in a total of 78 of 511 (15·3 %) blood samples that were tested and HI titres ranged from 20 to 5120 HIU per 50 µl. All birds with HI antibody were negative on feather HA testing (kappa = –0·178; P<0·0001). No evidence of any antigenic serotypes was detected by HI cross-reactivity studies using feather and blood samples from different individual psittacine bird species.

In the peach-faced lovebird flock, 47 out of 56 (83·9 %) birds were PCR positive on blood samples, but only 10 of these blood-PCR-positive birds (17·9 % prevalence) were also PCR positive on feather samples (Table 5). No bird was PCR feather positive without being PCR blood positive. Of the 10 blood-PCR-positive birds, five had detectable feather HA titres, ranging from 20 to 40 960 HAU per 50 µl. Five birds that were PCR positive on both blood and feathers had no detectable feather HA. The 56 birds had a low seroprevalence (16 %) and, of the nine birds that had detectable HI antibody titres (ranging from 20 to 320 HIU per 50 µl), none were feather HA or PCR positive but six were PCR positive on blood samples.


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Table 5. Test results obtained from 56 peach-faced lovebirds at a pet shop

 
Phylogenetic analysis of BFDV isolates
In this study, the ORF V1 sequences from nine new isolates of BFDV were determined with samples from five rainbow lorikeets (Trichoglossus haematodus), a red-collared lorikeet (T. haematodus rubritorquis), a musk lorikeet and two swift parrots (Table 1). Pairwise analysis of the sequences demonstrated that they were 88–99 % similar at the nucleotide level and 87–98 % similar at the amino acid level to each other and to other published sequences. Phylogenetic tree analysis demonstrated a clustering of all the lorikeet isolates and one of the swift parrot isolates with all other BFDV isolates obtained from species of the Loriidae. The second swift parrot isolate was a unique genotype closely related to the loriid group (Fig. 1). To confirm the sequences of the two swift parrot isolates, DNA extraction, cloning and sequencing was repeated. The swift parrots were young wild birds that died with acute PBFD shortly after being taken by National Parks rangers from a nest on Bruny Island (near Tasmania).



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Fig. 1. Neighbour-joining tree of 45 BFDV ORFV1 nucleotide sequences rooted to the Rep gene of canary circovirus. Numbers at the nodes indicates percentage bootstrap support. Putative loriid and cockatoo clades are shown with outlying cockatoo (*) and swift parrot ({dagger}) isolates highlighted.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The results indicate that PCR testing is more sensitive and specific than HA for detecting BFDV in feathers, because it detected evidence of viral DNA in feather suspensions that were HA negative (<20 HAU per 50 µl), even though the analytical limit of detection of BFDV by HA was better (1 log10) than PCR. This discrepancy is probably due to the enhanced process of extracting viral DNA from infected feather material in the absence of completed virus replication and release in feather dander. HA testing does provide a quantifiable indication of virus excretion and, when used in conjunction with PCR, provides a valuable internal control mechanism for the interpretation of results. From a diagnostic view point it is advantageous to know both results.

The presence of HI antibody in blood samples was inversely related to the presence of feather HA excretion and, except for one result, was also highly correlated with a negative feather PCR result. When used together, all three tests can provide useful information about the BFDV infection and immune status of a bird. Feather HA titres in excess of 640 HAU per 50 µl, particularly in cockatoos (Raidal et al., 1993a), are highly correlated with the presence of chronic disease and have been used to confirm a clinical diagnosis of PBFD. Problems with non-specific HA reactions have not previously been reported in feather samples but are relatively common in faecal samples (Raidal et al., 1993a). Confirmation of results by inhibition of the HA activity with BFDV-specific antibody is therefore recommended and should form part of a standard operating procedure. Parallel tests using BFDV-sensitive and BFDV-insensitive erythrocytes can also be used to ensure that the observed haemagglutination is specific and not due to other antigens (Raidal & Cross, 1994a).

The HI test has been used for seroepidemiological studies of BFDV infection in wild and captive birds (Raidal et al., 1993b; Raidal & Cross, 1994b) and the presence of HI antibody titres is a strong negative predictive indicator for PBFD (Raidal et al., 1993a; Ritchie et al., 1991), but birds with active or persistent BFDV infection may have low anti-BFDV HI titres that wax and wane. The non-detectable and low HI titres that occur in PBFD-affected birds may be explained by the severe damage that occurs to the bursa and thymus and or by the apparently persistent infections that occur in macrophages (Latimer et al., 1991).

Interpretation of any BFDV infection diagnostic testing regime must consider the signalment, clinical signs and history of the bird and its environment. HA and HI assays are quantitative and provide valuable laboratory information that can influence clinical decisions, but sources of suitable erythrocytes can be limited and differences in the agglutinating ability of erythrocytes obtained from different individuals of the same species have been reported (Sanada & Sanada, 2000). However, this is insufficient reason alone to discount HA as a diagnostic assay. As in any diagnostic assay, standardized procedures and appropriate internal controls should be used to provide reliable and valid results. Nevertheless, there is a need to develop other methods for quantifying BFDV excretion in feathers and faeces, because such information can be very important for guiding diagnostic judgements. Real-time PCR assays for BFDV infection may provide this information (Raue et al., 2004) but such techniques do so by detecting viral DNA and not antigen and their interpretation, from a clinical perspective, may not necessarily be any better than conventional non-quantitative PCR methods.

There has been only one report of the development of a direct ELISA for detecting anti-BFDV antibodies in psittacine bird sera (Johne et al., 2004), but this method of testing has yet to be validated with a large number of samples from birds with known health status. HI assay is likely to remain the gold standard for anti-BFDV antibody detection for several reasons. The main advantage of the HI antibody detection system is that a secondary antibody directed against psittacine IgY is not required, as is necessary in a direct ELISA. Johne et al. (2004) used a truncated recombinant capsid protein as the antigen in their ELISA and only 11 serum samples from seven psittacine bird species were tested and the secondary antibody was raised against IgY from an African grey parrot. There have been only limited studies of the cross-reactivity of anti-psittacine IgY antibody preparations, so one could never be certain whether serum from a rare species that tested negative was truly negative or whether the secondary antibody failed to recognize immunoglobulin from that particular species. Within the Cacatuidae, there are six genera including 21 species and, within the Psittacidae, there are 78 genera including 332 species. This present paper and others have shown that HI testing is suitable for detecting anti-BFDV antibodies in sera from a large proportion of these 353 species. The use of a truncated recombinant protein in an ELISA might also limit the assay's specificity. Until these issues are resolved, HI will probably continue to be the most reliable test for detecting BFDV antibodies.

The high blood PCR prevalence (83·9 %) and low seroprevalence (16 %) detected in the flock of peach-faced lovebirds (Agapornis roseicollis) (Table 5) could be explained by the flock being recently infected following mixing of birds from different sources at the pet shop. Seroprevalences have been shown to be much higher in endemically infected flocks of Agapornis sp. (62 %) and cockatoos (41–94 %) (Raidal & Cross, 1994b). Alternatively, there may be a high prevalence of latent or chronic carrier BFDV infections in Agapornis spp. Nevertheless, our observations that PCR can be more sensitive with blood versus feather samples is in contrast to that of Hess et al. (2004), who found a much higher prevalence of BFDV DNA in feather samples collected from budgerigars (Melopsittacus undulatus) even though there was poor correlation between PCR results and the presence or absence of feather lesions. Perhaps the reason for the higher prevalence of BFDV DNA in lovebirds compared with budgerigars could be explained by biological or immunological factors of the host species.

It is also important to consider that PCR tests may vary in sensitivity and specificity between laboratories, even when the same primers and optimization methods are employed (East et al., 2004). Our PCR prevalence data are similar to those reported by Bert et al. (2005) but much lower compared with those reported by Rahaus & Wolff (2003), who found a much higher prevalence (39 %) of BFDV DNA in feather samples collected from 146 clinically normal psittacine birds and even non-psittacine birds in Germany. Non-specific amplification of other avian circovirus amplicons was mooted as one possible reason for the latter observation but, in our experience, the primers designed by Ypelaar et al. (1999) do not amplify product from samples known to contain non-psittacine avian circoviruses. PCR assays for infectious agents have a theoretical high sensitivity and specificity but in practice they are rarely 100 % sensitive or specific (East et al., 2004; Peter et al., 2000; Muller-Doblies et al., 1998) and may even be only slightly more sensitive than conventional methods for virus detection (Mochizuki et al., 1993). Nested PCR assays can increase the sensitivity of an assay but the extra level of complexity can undo any gains in sensitivity or interfere with test specificity. False-negative PCR results may occur when inhibitors such as heparin (Holodniy et al., 1991) or biological materials in samples interfere with the assay (Konet et al., 2000) or as a result of laboratory operator error. False-positive results can occur with cross-contamination during sample collection or with laboratory handling and it is well accepted for other viruses that a positive PCR test result, on its own, is not a demonstration of active viral infection, as non-replicating DNA may take up to 3 months to clear from blood (Lazizi & Pillot, 1993), and it is for this reason that PCR-positive birds without clinical signs should be recommended for retesting after 3 months (Dahlhausen & Radabaugh, 1993).

PCR technology should be used together with, and not replace, conventional diagnostic testing for PBFD (Cross, 1996). The data presented in this present paper indicate merit in having a two-stage method for BFDV sample testing. In our experience, HA testing provides a valuable second method for identifying those birds that may be chronically affected and excreting large amounts of virus in feather dander versus those birds that may only be recently infected and not shedding virus but mounting an effective immune reaction.

Ypelaar et al. (1999) found that consistent PCR results could only be achieved with primers designed to amplify ORF V1, which encodes the Rep protein and thus is more likely than the capsid protein to be genetically conserved. However, because of the diversity of BFDV genotypes, PCR-based technologies may not detect all isolates even when conserved primers are used (Heath et al., 2004; Bassami et al., 2001; Ritchie et al., 2003; Johne et al., 2004). This is another reason for having a two-stage testing regime to capture isolates that may be genetically unique but still capable of causing haemagglutination. However, we found no evidence of this possibility in our sample set and the five false-positive HA reactors that we detected were cleared by filtering the sample through 0·22 µm filters and were not inhibited by anti-BFDV antibody.

There has been debate in the literature over the existence of a BFDV strain genetically adapted to lorikeets and parrots (Ritchie et al., 2003; Raue et al., 2004; de Kloet & de Kloet, 2004; Heath et al., 2004) and the evolution of species-specific BFDV genotypes such as cockatoo, budgerigar, lorikeet and lovebird lineages. This was the reason why we determined the DNA sequences of the isolates we obtained from lorikeets and the two swift parrot isolates. Swift parrots are an endangered species belonging to the family Psittacidae (Christidis et al., 1991; Christidis & Boles, 1995) but are behaviourally and anatomically similar to lorikeets, which justified studying the BFDV isolates obtained from these two birds. We compared the generated DNA sequence data with 36 previously described ORF V1 BFDV sequences from psittacine birds from Australia, USA, UK, Germany, South Africa, Portugal, Austria and New Zealand. The sequences were similar (86–97 %) at the nucleotide level and, with the exception of one swift parrot isolate (isolate 3-SP-TS), our results shown in an inferred phylogenetic tree (Fig. 1) rooted to canary circovirus are supportive of the clustering of BFDV isolates from lorikeets and lories into a loriid genotype first proposed by Ritchie et al. (2003). There is a relatively high degree of genetic diversity in BFDV and, as more sequence data become available, the emergence of genotypes obtained from species of the Psittacidae is not unexpected given the larger number of extant bird species in this family. However, the biological significance of BFDV genotype clades is unknown. Until transmission studies prove otherwise, it must continue to be assumed that all psittacine bird species are potentially susceptible to each genotype; indeed, the putative high degree of recombination events within ORF V1 supports this assumption (Heath et al., 2004).

There is evidence that recombination might contribute substantially more to genetic variation than genetic drift within ORF V1, and this can result in inaccurate phylogenetic inferences (Heath et al., 2004). However, there is little proof of multiple BFDV isolate infections within psittacine hosts to permit such recombination events. The two sequences we obtained from swift parrots are good evidence that different isolates can at least naturally infect siblings within the same nest hollow. DNA sequence data from our two swift parrot isolates suggest that this species is naturally susceptible to both loriid and psittacid genotypes, which would be consistent with cross-infection of BFDV between lorikeets and swift parrots. Swift parrots are a monotypic genus that probably evolved in the south-east of Australia from a granivorous psittacid into a specialist nectarivorous bird before the more recent introduction of trichoglossid lorikeets (Christidis et al., 1991). It competes closely for nectar and pollen as well as nesting sites with several lorikeet and parrot species (Gartrell & Jones, 2001) including, at Bruny Island, musk lorikeets, eastern rosellas (Platycercus eximius) and green rosellas (Platycercus caledonicus). Swift parrots use different nest holes each year according to the proximity of flowering trees (Dr Brett Gartrell, personal communication). The wild swift parrot population currently consists of fewer than 1300 breeding pairs and is thought to be decreasing by more than 1 % every year. Subclinical BFDV infections are well known in wild rainbow and scaly-breasted lorikeets in Australia which rarely develop chronically progressive lesions characteristic of PBFD in cockatoos, but evidence that this is solely due to less virulent genotypes as suggested by Raue et al. (2004) rather than host defence factors is yet to be resolved. Such lorikeets pose a unique problem in that birds with clinical disease are frequently rescued and rehabilitated by wildlife carers in the eastern states of Australia, which may promote the spread of BFDV carriers in the wild. Our results provide the first evidence that BFDV isolates derived from lorikeets may be able to infect other psittacine bird species.


   ACKNOWLEDGEMENTS
 
Funding for this research was provided by grants from the National Heritage Trust and Murdoch University. The PhD scholarship of B. K. was funded by the Iranian Ministry of Science, Investigation and Technology and the Ministry of Agriculture of Jahad. We would also like to thank Ian Robertson and Douglas Bearham for statistical and phylogenetic advice, respectively.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 22 June 2005; accepted 12 August 2005.



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