小太阳鹦鹉禽多瘤病毒感染病例分析

目的确诊某宠物鹦鹉饲养场的小太阳鹦鹉幼雏发生大规模发病死亡疫情的病原.方法对送检的发病幼雏进行临床剖检及病理组织学观察,取其肝脏组织提取DNA进行禽多瘤病毒、喙羽病病毒、腺病毒核酸检测.结果发病鹦鹉均以肝坏死、肠道出血等消化系统病变为主,胸腺、法氏囊等免疫器官也有一定程度病理损伤.病原检测结果显示所有发病鹦鹉均为禽多瘤病毒阳性,喙羽病病毒、腺病毒阴性;对PCR扩增产物测序后分析显示,该分离株与台湾流行的鹦鹉禽多瘤病毒同源性最高.结论该病例的发现提示我国应加大对宠物鹦鹉禽多瘤病毒的检测力度,防止输入性感染,鹦鹉饲养场应进行禽多瘤病毒净化,并采取有效的生物安全措施防止带毒鹦鹉的引进.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

An outbreak of Pacheco's parrot disease in a psittacine bird collection and an attempt to control it by vaccination

An outbreak of Pacheco's parrot disease (PPD) is described in a psittacine bird collection of a municipal ornithological garden. A herpes virus was isolated and judged to be the causative agent from the mortality recorded. A formaldehyde/heat-inactivated experimental vaccine was prepared from polyethyleneglycol purified virus. The vaccine was locally and systemically well tolerated by all birds. Serocon-version was demonstrated by neutralization tests in most of the vaccinated birds of the genera Ara, Anodorhynchus and Probosciger.     

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.     

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.     

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'.     

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.     

Herpesvirus infection resembling Pacheco's disease in Amazon parrots

Herpesvirus infection was diagnosed in two parrots that died suddenly in a pet shop. Gross lesions were confined to reddening of the small intestinal mucosa. Histological examination of tissues from one specimen revealed a hepatitis and splenitis accompanied by intranuclear inclusions. Cowdry type A inclusions were observed in tissue culture and within the nuclei of chick embryo hepatocytes. Virus particles with a morphological similarity to herpesviruses were identified in electron microscope studies. The condition was considered to represent a case of Pacheco's parrot disease.     

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.     

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.     

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.     

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.