Detection of Immunoreactivity to Psittaciform Bornavirus in the Serum of a Wild Cacatuid in Victoria, Australia.

An indirect immunofluorescence serologic assay, PCR assay, and histopathology were used to screen for psittaciform orthobornaviruses (PaBV) in wild Cacatuidae in Victoria, Australia. Anti-PaBV antibodies were detected, but PCR and histopathology did not detect PaBV. This study presents the first evidence of PaBV in wild birds in Australia.

Age-dependent development and clinical characteristics of an experimental parrot bornavirus-4 (PaBV-4) infection in cockatiels (Nymphicus hollandicus).

Parrot bornavirus (PaBV) is a pathogen often found in psittacine populations. Infected, clinically healthy carrier birds are of major importance for epidemiology, but the underlying pathomechanism of this carrier status is poorly understood. The age, implying the maturation status of the immune system, at the time of infection might be significant for the clinical outcome. Therefore, two groups of 11 cockatiels of different ages (adult and newly hatched) were inoculated with a PaBV-4 isolate intravenously. The trial lasted for 233 days and all birds were observed for clinical signs, PaBV-RNA shedding and anti-PaBV antibody production. At the end of the trial, histopathology, immunohistochemistry, PCR and virus re-isolation were performed. All 22 birds seroconverted and shed PaBV-RNA during the investigation period; the juvenile group earlier and more homogeneously. Nine of 11 birds of the adult group developed clinical signs; five birds died or had to be euthanized before the end of the study. In the juvenile group none of the birds developed clinical signs and only one bird died due to bacterial septicaemia. Eight birds of the adult group, but none of the juvenile group, showed a dilatation of the proventriculus. PaBV-RNA detection and virus re-isolation were successful in all birds. Immunohistochemically, PaBV antigen was found in all birds. Histopathology revealed mononuclear infiltrations in organs in birds of both groups, but the juveniles were less severely affected in the brain.Thus, PaBV infection at an age with a more naïve immune system makes the production of carrier birds more likely.RESEARCH HIGHLIGHTS PaBV infection at a young age might favour the development of carrier birds.Cockatiels infected at a very young age showed inflammation but no clinical signs.The juvenile group started seroconversion and PaBV-RNA shedding earlier.Seroconversion and PaBV-RNA shedding occurred more homogeneously in the juveniles.

Studies on immunity and immunopathogenesis of parrot bornaviral disease in cockatiels.

We have demonstrated that vaccination of cockatiels (Nymphicus hollandicus) with killed parrot bornavirus (PaBV) plus recombinant PaBV-4 nucleoprotein (N) in alum was protective against disease in birds challenged with a virulent bornavirus isolate (PaBV-2). Unvaccinated birds, as well as birds vaccinated after challenge, developed gross and histologic lesions typical of proventricular dilatation disease (PDD). There was no evidence that vaccination either before or after challenge made the infection more severe. Birds vaccinated prior to challenge largely remained free of disease, despite the persistence of the virus in many organs. Similar results were obtained when recombinant N, in alum, was used for vaccination. In some rodent models, Borna disease is immune mediated thus we did an additional study whereby cyclosporine A was administered to unvaccinated birds starting 1day prior to challenge. This treatment also conferred complete protection from disease, but not infection.

Beak and feather disease virus carriage by Knemidocoptes pilae in a sulphur-crested cockatoo (Cacatua galerita).

BACKGROUND: This paper describes the pathology associated with psittacine beak and feather disease in a wild sulphur-crested cockatoo with concurrent knemidocoptic mange, cestodiasis and mycotic encephalitis. METHODS & RESULTS: Large numbers of Knemidocoptes pilae Lavoipierre and Griffiths, 1951 (Acari: Epidermoptidae, Knemidokoptinae) were identified in affected skin associated with enhanced expression of beak and feather disease virus (BFDV) determined by immunohistochemistry. Also, BFDV antigen was demonstrated in high concentration in the gut and faecal sacs of mites, raising the possibility of ectoparasites as fomites and vectors of BFDV transmission. Large numbers of Raillietina spp. cestodes were present in the intestines. Within the brain there was a focally extensive region of necrosis and inflammation associated with branching, septate, pigmented hyphae consistent with zygomycete fungal infection. CONCLUSION: This case highlights the potential immunosuppressive effects of BFDV infection and its potential as a keystone pathogen in the Australian environment.

From nerves to brain to gastrointestinal tract: A time-based study of parrot bornavirus 2 (PaBV-2) pathogenesis in cockatiels (Nymphicus hollandicus).

Parrot bornaviruses (PaBVs) are the causative agents of proventricular dilatation disease, however key aspects of its pathogenesis, such as route of infection, viral spread and distribution, and target cells remain unclear. Our study aimed to track the viral spread and lesion development at 5, 10, 20, 25, 35, 40, 60, 80, 95 and 114 dpi using histopathology, immunohistochemistry, and RT-PCR. After intramuscular inoculation of parrot bornavirus 2 (PaBV-2) in the pectoral muscle of cockatiels, this virus was first detected in macrophages and lymphocytes in the inoculation site and adjacent nerves, then reached the brachial plexus, centripetally spread to the thoracic segment of the spinal cord, and subsequently invaded the other spinal segments and brain. After reaching the central nervous system (CNS), PaBV-2 centrifugally spread out the CNS to the ganglia in the gastrointestinal (GI) system, adrenal gland, heart, and kidneys. At late points of infection, PaBV-2 was not only detected in nerves and ganglia but widespread in the smooth muscle and/or scattered epithelial cells of tissues such as crop, intestines, proventriculus, kidneys, skin, and vessels. Despite the hallmark lesion of PaBVs infection being the dilation of the proventriculus, our results demonstrate PaBV-2 first targets the CNS, before migrating to peripheral tissues such as the GI system.

Viral vector vaccines expressing nucleoprotein and phosphoprotein genes of avian bornaviruses ameliorate homologous challenge infections in cockatiels and common canaries.

Avian bornaviruses are causative agents of proventricular dilatation disease (PDD), an often fatal disease of parrots and related species (order Psittaciformes) which is widely distributed in captive psittacine populations and may affect endangered species. Here, we established a vaccination strategy employing two different well described viral vectors, namely recombinant Newcastle disease virus (NDV) and modified vaccinia virus Ankara (MVA) that were engineered to express the phosphoprotein and nucleoprotein genes of two avian bornaviruses, parrot bornavirus 4 (PaBV-4) and canary bornavirus 2 (CnBV-2). When combined in a heterologous prime/boost vaccination regime, NDV and MVA vaccine viruses established self-limiting infections and induced a bornavirus-specific humoral immune response in cockatiels (Nymphicus hollandicus) and common canaries (Serinus canaria forma domestica). After challenge infection with a homologous bornavirus, shedding of bornavirus RNA and viral loads in tissue samples were significantly reduced in immunized birds, indicating that vaccination markedly delayed the course of infection. However, cockatiels still developed signs of PDD if the vaccine failed to prevent viral persistence. Our work demonstrates that avian bornavirus infections can be repressed by vaccine-induced immunity. It represents a first crucial step towards a protective vaccination strategy to combat PDD in psittacine birds.

Parrot Bornavirus (PaBV)-2 isolate causes different disease patterns in cockatiels than PaBV-4.

Psittaciform 1 bornavirus (PaBV) has already been shown to be the aetiologic agent of proventricular dilatation disease, a significant disease of birds. However, the pathogenesis of PaBV infection has not yet been resolved and valid data regarding the pathogenicity of different PaBV species are lacking. Thus, the present study was aimed to characterize the influence of two different PaBV species on the course of disease. Eighteen cockatiels were inoculated intracerebrally (i.c.) or intravenously (i.v.) with a PaBV-2 isolate under the same conditions as in a previous study using PaBV-4. Birds were surveyed and sampled for 33 weeks to analyse the course of infection and disease in comparison to that of PaBV-4. Similar to PaBV-4, PaBV-2 induced a persistent infection with seroconversion (from day 6 p.i. onwards) and shedding of viral RNA (from day 27 p.i. onwards). However, in contrast to PaBV-4, more birds displayed clinical signs and disease progression was more severe. After PaBV-2 infection, 12 birds exhibited clinical signs and 10 birds revealed a dilated proventriculus in necropsy. After PaBV-4 infection only four birds revealed clinical signs and seven birds showed a dilatation of the proventriculus. Clinically, different courses of disease were observed after PaBV-2 infection, mainly affecting the gastrointestinal tract. This had not been detected after PaBV-4 infection where more neurological signs were noted. The results provide evidence for different disease patterns according to different PaBV species, allowing the comparison between the infection with two PaBV species, and thus underlining the role of viral and individual host factors for disease outcome.

Phylogeny of beak and feather disease virus in cockatoos demonstrates host generalism and multiple-variant infections within Psittaciformes.

Phylogenetic analyses of the highly genetically diverse but antigenically conserved, single-stranded circular, DNA genome of the avian circovirus, beak and feather disease virus (BFDV) from cockatoo species throughout Australia demonstrated a high mutation rate for BFDV (orders of magnitude fall in the range of 10(-4) substitutions/site/year) along with strong support for recombination indicating active cross-species transmission in various subpopulations. Multiple variants of BFDV were demonstrated with at least 30 genotypic variants identified within nine individual birds, with one containing up to 7 variants. Single genetic variants were detected in feathers from 2 birds but splenic tissue provided further variants. The rich BFDV genetic diversity points to Australasia as the most likely geographical origin of this virus and supports flexible host switching. We propose this as evidence of Order-wide host generalism in the Psittaciformes characterised by high mutability that is buffered by frequent recombination and slow replication strategy.

No contact transmission of avian bornavirus in experimentally infected cockatiels (Nymphicus hollandicus) and domestic canaries (Serinus canaria forma domestica).

Avian bornaviruses (ABV) are the causative agents of proventricular dilatation disease (PDD), a widely distributed disease of parrots. Distinct ABV lineages were also found in various non-psittacine avian species, such as canaries, but the pathogenic role of ABV in these species is less clear. Despite the wide distribution of ABV in captive parrots and canaries, its mode of transmission is poorly understood: both horizontal transmission via the urofaecal-oral route and vertical transmission are discussed to play a role. In this study we investigated pathology and horizontal transmission of ABV in domestic canaries (Serinus canaria forma domestica) and cockatiels (Nymphicus hollandicus), two natural host species commonly used for experimental ABV infections. ABV inoculation resulted in persistent infection of all inoculated animals from both species. ABV-infected cockatiels exhibited PDD-like symptoms, such as neurologic signs or shedding of undigested seeds. In contrast, infected domestic canaries did not develop clinical disease. Interestingly, we did not detect viral RNA in cloacal swabs and organ samples or ABV-specific antibodies in serum samples of contact-exposed sentinel birds from either species at any time during a four months observation period. Our results strongly indicate that horizontal transmission of ABV by direct contact is inefficient in immunocompetent fully fledged domestic canaries and cockatiels.

Differential expression of two isolates of beak and feather disease virus capsid protein in Escherichia coli.

Expression of recombinant beak and feather disease virus (BFDV) capsid-associated protein (Cap) has relied on inefficient techniques that typically produce low yields or use specialized expression systems, which greatly increase the cost and expertise required for mass production. An Escherichia coli system was used to express recombinant BFDV Cap derived from two isolates of BFDV, from a Long-billed Corella (Cacatua tenuirostris) and an Orange-bellied parrot (OBP; Neophema chrysogaster). Purification by affinity and size exclusion chromatography was optimized through an iterative process involving screening and modification of buffer constituents and pH. A buffer containing glycerol, ß-mercaptoethanol, Triton X-100, and a high concentration of NaCl at pH 8 was used to increase solubility of the protein. The final concentration of the corella-isolated BFDV protein was fifteen- to twenty-fold greater than that produced in previous publications using E. coli expression systems. Immunoassays were used to confirm the specific antigenicity of recombinant Cap, verifying its validity for use in continued experimentation as a potential vaccine, a reagent in diagnostic assays, and as a concentrated sample for biological discoveries.

Efficient isolation of avian bornaviruses (ABV) from naturally infected psittacine birds and identification of a new ABV genotype from a salmon-crested cockatoo (Cacatua moluccensis).

Avian bornaviruses (ABV) have been discovered in 2008 as the causative agent of proventricular dilatation disease (PDD) in psittacine birds. To date, six ABV genotypes have been described in psittacines. Furthermore, two additional but genetically different ABV genotypes were recognized in non-psittacine birds such as canary birds and wild waterfowl. This remarkable genetic diversity poses a considerable challenge to ABV diagnosis, since polymerase chain reaction (PCR) assays may fail to detect distantly related or as yet unknown genotypes. In this study we investigated the use of virus isolation in cell culture as a strategy for improving ABV diagnosis. We found that the quail fibroblast cell line CEC-32 allows very efficient isolation of ABV from psittacine birds. Isolation of ABV was successful not only from organ samples but also from cloacal and pharyngeal swabs and blood samples collected intra vitam from naturally infected parrots. Importantly, using this experimental approach we managed to isolate a new ABV genotype, termed ABV-7, from a salmon-crested cockatoo (Cacatua moluccensis). Phylogenetic analysis showed that ABV-7 is most closely related to the psittacine genotypes ABV-1, -2, -3, and -4 and clearly distinct from genotypes ABV-5 and -6. Our successful identification of ABV-7 emphasizes the necessity to consider the high genetic diversity when trying to diagnose ABV infections with high reliability and further shows that classical virus isolation may represent a useful diagnostic option, particularly for the detection of new ABV genotypes.

Molecular genetic and pathogenic characterization of psittacid herpesvirus type 1 isolated from a captive galah (Eolophus roseicapillus) in Japan.

Psittacid herpesvirus type 1 (PsHV-1) was isolated from a captive galah (Eolophus roseicapillus) in Japan that was suspected of having Pacheco's disease (PD), an acute fatal disease in psittacine birds. PsHV-1 has been classified into four genotypes based on the UL16 gene sequence. In the present study, we investigated the genetic and pathogenic characteristics of the isolated virus, FOY-1, compared with a reference strain, RSL-1. The FOY-1 strain was classified into PsHV-1 genotype 2. The FOY-1 strain was found to be less pathogenic to budgerigars than RSL-1, which was classified as genotype 4 in an in vivo study. This is the first report regarding the classification of originally isolated PsHV-1 in Japan and its characterization by animal infection experiment.

Unusual and severe lesions of proventricular dilatation disease in cockatiels (Nymphicus hollandicus) acting as healthy carriers of avian bornavirus (ABV) and subsequently infected with a virulent strain of ABV.

A flock of 14 apparently healthy cockatiels, purchased from a single aviary, was tested for the presence of avian bornavirus (ABV). Twelve birds were found to be intermittently shedding ABV, predominantly genotype 4. Four of the cockatiels known to be shedding ABV4 were subsequently challenged with the tissue culture derived, virulent M24 strain of ABV4. The challenged birds remained in apparent good health until day 92 when one was found dead. The remaining three birds began to exhibit severe neurologic signs, ataxia and convulsions on day 110 and were euthanized. On necropsy, all four birds showed mild proventricular enlargement. In contrast, histopathological examination showed unusually severe and widespread tissue lesions. These included massive lymphocytic infiltration and lymphoid nodule formation within and around the ganglia throughout the gastrointestinal tract. There were similar lesions in the medullary cords of the adrenal gland, heart, spleen, liver, kidney, lungs, pancreas, testes and ovary. Immunohistochemistry demonstrated ABV P antigen not only in the cells of the central and autonomic nervous systems, but also within the mononuclear cells infiltrating the various organs. Two healthy cockatiels, one of which was a known ABV carrier, were inoculated with uninfected tissue culture cells and euthanized on day 150. These birds showed no gross lesions of proventricular dilatation disease but had a mild lymphocytic infiltration in their liver, spleen, and kidneys. Prior infection with ABV did not therefore confer significant immunity on these birds, and may have resulted in increased disease severity following challenge.

Genetic analysis of beak and feather disease virus derived from a cockatiel (Nymphicus hollandicus) in Japan.

Psittacine beak and feather disease (PBFD), which is caused by beak and feather disease virus (BFDV), has been reported in a wide range of psittacine species, except the cockatiel (Nymphicus hollandicus), in which PBFD has rarely been reported. We detected BFDV from a case of PBFD in a cockatiel in the present study. The virus was designated CO-JA. The whole genome sequence of CO-JA had from 86 to 98% homology with BFDVs in psittacine species. CO-JA clustered with isolates derived from other cockatoos in phylogenetic analyses based on two major virus proteins. We concluded that genetic data cannot explain the reason why PBFD is rarely found in the cockatiel.

A blocking ELISA for the detection of antibodies to psittacine beak and feather disease virus (BFDV).

Currently, the only diagnostic test available routinely for the sero-diagnosis of BFDV is the haemagglutination-inhibition (HI) assay. This test, whilst useful and applicable to samples from a wide range of psittacine birds, is not an ideal assay; it requires erythrocytes from live animals, virus purified from the feathers of infected birds and polyclonal antibody preparations in order to perform the assay. Variations in these reagents make consistency between tests difficult to achieve, underscoring the need for a new test with standardised reagents for the sero-diagnosis of BFDV infection which has led to the development of an antibody response. The methods used to develop a novel "blocking" (or "competitive") ELISA (bELISA) for the detection of anti-BFDV antibodies in psittacine sera are presented in this paper. The assay was developed using a baculovirus-expressed recombinant BFDV capsid protein and a newly developed monoclonal antibody raised against this protein. The assay was then validated with 160 samples from eastern long-billed corellas (Cacatua tenuiostris) vaccinated with the recombinant capsid protein and challenged with live virus and samples from 82 cockatiels known to be HI negative. The bELISA described in this study is a sensitive and specific diagnostic test and should have wide application for the sero-diagnosis of BFDV.

Assessment of recombinant beak and feather disease virus capsid protein as a vaccine for psittacine beak and feather disease.

Beak and feather disease virus (BFDV) is a significant pathogen of wild Australasian and African psittacine birds. We assessed the immunogenicity of recombinant BFDV capsid (recBFDVcap) to protect against the development of psittacine beak and feather disease (PBFD). Long-billed corellas (Cacatua tenuirostris) (n=13) received (by injection) 1 ml vaccine containing 10 microg recBFDVcap on day 0 and 0.4 ml vaccine containing 66.8 microg recBFDVcap on day 11. All vaccinated corellas and five non-vaccinated control corellas were given 0.4 ml BFDV suspension [titre=log(2) 12 haemagglutination units (HAU) 50 microl(-1)] intramuscularly and 0.1 ml orally 16 days after booster vaccination. Blood was collected during the vaccination period and blood and feathers were collected after BFDV administration. Testing of blood samples included BFDV DNA detection by PCR and quantitative PCR (qPCR) as well as antibody detection by haemagglutination inhibition (HI) and on feather samples, BFDV DNA and antigen was detected by haemagglutination (HA) and qPCR. Four of 97 blood samples collected from vaccinated birds after virus challenge tested positive by PCR, whereas 17 of 35 samples taken from non-vaccinated control corellas tested positive. Vaccinated birds did not develop feather lesions, had only transient PCR-detectable viraemia and had no evidence of persistent infection 270 days post-challenge using PCR, histopathology and immunohistochemistry. Non-vaccinated control corellas developed transient feather lesions and had PCR, HI and HA test results consistent with PBFD. They were BFDV PCR-positive for up to 41 days post-challenge and qPCR demonstrated reduced virus replication in vaccinated birds compared with non-vaccinated control birds.

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.

Identification of a natural multi-recombinant of Newcastle disease virus.

Newcastle disease (ND), caused by ND virus (NDV), is one of the most serious illnesses of birds, particularly chickens, and has been one of the major causes of economic losses in the poultry industry. Live vaccines are widely used to prevent chicken from NDV all over the world. Given the implications that recombination has for RNA virus evolution, it is clearly important to determine the extent to which recombination plays a role in NDV evolution. In this study, we performed the phylogenetic and recombination analysis on complete NDV genomes. A natural multi-recombinant cockatoo/Indonesia/14698/90 (AY562985) was identified. Its two minor parental-like strains might be from the NDV vaccine lineage and anhinga/U.S.(Fl)/44083/93 lineage, respectively. Our study suggests that recombination plays a role in NDV evolution. Especially, the study also suggests that live vaccines have capacity to play roles in shaping NDV evolution by homologous recombination with circulating virus.

Endocrine pancreatic insufficiency secondary to chronic herpesvirus pancreatitis in a cockatiel (Nymphicus hollandicus).

A cockatiel (Nymphicus hollandicus) examined because of weight loss, polydipsia, and polyuria was diagnosed with diabetes mellitus based on the presence of glucosuria and marked hyperglycemia. Medical attempts to manage the diabetes mellitus were unsuccessful, and the bird was euthanatized. Histopathologic examination of the pancreas revealed a chronic active pancreatitis with herpesviral inclusions in many of the pancreatic acinar and duct cells. Psittacid herpesvirus-1 (PsHV-1) DNA was amplified from the lesion by polymerase chain reaction. Sequencing of the amplicon showed it to be the genotype 1 variant, which is most commonly associated with Pacheco's disease, an acute rapidly fatal systemic infection. The findings in this case suggest that the PsHV-1 genotype may also cause a localized disease of the pancreas. Infection with this virus should be considered as a differential diagnosis in birds with pancreatitis with or without diabetes mellitus.

Psittacine circovirus infection in parakeets of the genus Eunymphicus and treatment with beta-(1,3/1,6)-D-glucan.

Eight captive-bred horned parakeets (Eunymphicus cornutus) and four captive-bred Major Mitchell cockatoos (Cacatua leadbeateri) from the same aviary tested positive for psittacine circovirus (PsCV) DNA in whole blood by nested-polymerase chain reaction (PCR). The chronic form of disease with feather fragility and loss was observed in three horned parakeets. Infection in other individuals was subclinical. Immunosuppression, either hematologically or as susceptibility to secondary infections, was not observed. Treatment consisted of the administration of beta-(1,3/1,6)-D-glucan from oyster mushroom (Pleurotus ostreatus). Excluding two accidentally dead parakeets, four out of the original six horned parakeets, and all Major Mitchell cockatoos were negative for PsCV DNA in whole blood in 7-9 mo after the treatment was started. Even though the absence of PsCV DNA in blood does not signify elimination of the virus from the whole organism, these preliminary results indicate a possible effect of beta-glucan in the treatment of PsCV infection. To the author's knowledge, this is the first report of PsCV in horned parakeets.