PARROT BORNAVIRUSES IN PSITTACINES KEPT IN CAPTIVITY IN THE STATE OF SANTA CATARINA, BRAZIL.

Parrot bornaviruses are responsible for proventricular dilatation disease (PDD) in psittacines. This study aimed to determine the occurrence and factors associated with Parrot bornaviruses infection in psittacines kept in captivity in a state in the southern region of Brazil. A cross-sectional study was carried out with 192 birds from two facilities (A and B) in 2019, using choanal, esophageal, and cloacal swabs and feathers, totaling 768 samples subjected to reverse-transcription polymerase chain reaction (RT-PCR), for the matrix (M) protein gene with a final product of 350 base pairs (bp). Genetic sequencing of three positive samples was performed by the Sanger method. In the study, the overall virus occurrence was 35.9% (69/192), with 40.4% (42/104) in Facility A and 30.7% (27/88) in Facility B. Sequencing analysis of the samples revealed the presence of Parrot bornavirus 2 (PaBV-2) in both facilities. Swab samples from the choanal (40/69), esophageal (30/69), cloacal (35/69), and feather (15/69) tested positive, facilitating the molecular diagnosis of Parrot bornaviruses. The results indicated that there is no single ideal sample type for antemortem molecular diagnosis of this virus. Simultaneously testing all four samples at the same time point yielded more diagnoses than testing any single sample among the four. Most of the 29 sampled psittacine species were native, and 46.9% of the birds (90/192) consisted of endangered species. Among the psittacines that tested positive, 88.4% (61/69) were clinically healthy, and 8.7% (6/69) exhibited clinical or behavioral signs, including behavioral changes, alterations in feathering, and changes in body score at the time of collection. This study showcases the application of minimally invasive sampling for diagnosing Parrot bornaviruses, enabling sample collection when the birds are restrained for clinical evaluation. This approach facilitates a prompt and effective antemortem diagnosis, thereby serving as an efficient screening method for parrots kept in captivity.

Surveillance of Parrot Bornavirus in Taiwan Captive Psittaciformes.

Parrot bornavirus (PaBV) is an infectious disease linked with proventricular dilatation disease (PDD) with severe digestive and neurological symptoms affecting psittacine birds. Despite its detection in 2008, PaBV prevalence in Taiwan remains unexplored. Taiwan is one of the leading psittacine bird breeders; hence, understanding the distribution of PaBV aids preventive measures in controlling spread, early disease recognition, epidemiology, and transmission dynamics. Here, we aimed to detect the prevalence rate of PaBV and assess its genetic variation in Taiwan. Among 124 psittacine birds tested, fifty-seven were PaBV-positive, a prevalence rate of 45.97%. Most of the PaBV infections were adult psittacine birds, with five birds surviving the infection, resulting in a low survival rate (8.77%). A year of parrot bornavirus surveillance presented a seasonal pattern, with peak PaBV infection rates occurring in the spring season (68%) and the least in the summer season (25%), indicating the occurrence of PaBV infections linked to seasonal factors. Histopathology reveals severe meningoencephalitis in the cerebellum and dilated cardiomyopathy of the heart in psittacine birds who suffered from PDD. Three brain samples underwent X/P gene sequencing, revealing PaBV-2 and PaBV-4 viral genotypes through phylogenetic analyses. This underscores the necessity for ongoing PaBV surveillance and further investigation into its pathophysiology and transmission routes.

Betanodavirus meningoencephalitis in an Atlantic blue marlin.

Viral nervous necrosis (viral encephalopathy and retinopathy) is caused by piscine nodavirus (Nodaviridae, Betanodavirus). Since 1986, this highly infectious virus has caused mass mortalities of up to 100% in farmed saltwater and freshwater fish around the world (with the exception of South America and Antarctica), affecting >60 species across 10 orders. The Atlantic blue marlin (Makaira nigricans Lacépède, 1802) is a top-level predator found throughout the tropical waters of the Atlantic and Indo-Pacific oceans. Despite their popularity as a sportfish, relatively little is known about the Atlantic blue marlin and other billfish. We describe here chronic betanodavirus infection in a juvenile Atlantic blue marlin, which is, to our knowledge, the first report of disease in M. nigricans.

Genetic trends in parrot Bornavirus: a clinical analysis.

Parrot Bornavirus (PaBV) has been reported to cause indigestion and other wasting symptoms such as weight loss and lethargy. The pathogenesis of PaBV has yet to be fully elucidated. This study reports PaBV infections in South Korea and suggests a trend in the genetic information gathered from clinical cases. A total of 487 birds with or without clinical symptoms were tested for bornavirus. Twelve of 361 asymptomatic birds tested positive for bornavirus, while 15 of 126 birds with various symptoms tested positive. A segment of approximately 1,540 bps including the N, X, P and M proteins were obtained from 23 of the positive strains and analyzed with other strains found on GenBank that had clinical information. PaBV was type 2 and 4 in South Korea, and certain amino acid sequences showed a difference between symptom presenting animals and asymptomatic animals in the X protein and P protein. When considering that some asymptomatic cases may have been latent infections at the time of examination, it is plausible these trends may grow stronger with time. Majority of PaBV was type 4 in South Korea. If these trends are confirmed, diagnosis of potentially pathogenic PaBVs in a clinical manner will be possible during the early stages of infection.

Surveillance for Avian Bornavirus in Colorado and Wyoming, USA, Raptor Populations.

Avian bornavirus (ABV) is known to infect at least 80 avian species and is associated with avian bornaviral ganglioneuritis (ABG). Avian bornaviral ganglioneuritis is characterized by a lymphoplasmacytic infiltration of the nervous tissue, mainly affecting the nerves that supply the gastrointestinal tract of birds. This disease is diagnosed commonly in psittacines under human care and has been demonstrated in wild bird species; however, its occurrence in raptors is largely unknown. Because of the commonality of ABV in the pet bird population, there is concern about the spread of this virus to other companion avian species, such as falconry birds, as well as wildlife. This prospective study used reverse-transcription quantitative PCR (RT-qPCR) to survey free-ranging Colorado and Wyoming, US, raptor populations for ABV. Quantitative PCR was performed on mixed conjunctival-choanal-cloacal swabs collected from live birds (n=139). In dead birds, a combination of mixed swabs (n=265) and tissue samples of the brain (n=258), heart (n=162), adrenal glands (n=162), liver (n=162), kidney (n=139), spinal cord (n=139), and brachial plexus (n=139) were evaluated. All 1,565 swab and tissue samples RT-qPCR results from the 404 birds evaluated were negative. Based on these results and a lack of clinical signs suggestive of ABG, ABV is likely not a prevalent pathogen in Colorado and Wyoming raptor populations at this time.

Molecular detection of bornavirus in parrots imported to China in 2022.

BACKGROUND: Avian bornavirus (ABV) is a neurotropic virus, it has been established as the primary causative agent of proventricular dilatation disease (PDD). However, substantial international trade and transnational trafficking of wild birds occur, potentially enabling these birds to harbor and transmit pathogens to domestic poultry, adversely affecting their well-being. Real-time RT-PCR was employed to detect the presence of PaBV-4 in parrots imported to China in 2022. RESULTS: In 2022, a total of 47 cloacal swabs from 9 distinct species of parrots were collected at the Wildlife Rescue Monitoring Center in Guangdong, China. The purpose of this collection was to detect the presence of PaBV-4. Using real-time PCR techniques, it was determined that the positive rate of PaBV-4 was 2.12% (1 out of 47) in parrots. The PaBV-4 virus was detected in a Amazona aestiva that had been adopted for one month. Conversely, all other species tested negative for the virus. Subsequently, the whole genome of the PaBV-4 GD2207 strains was sequenced, and the homology and genetic evolution between these strains and previously published PaBV-4 strains on GenBank were analyzed using DNAStar and MEGA7.0 software. The findings revealed that the full-length genome of PaBV-4 consisted of 8915 nucleotides and encoded six proteins. Additionally, it exhibited the highest nucleotide similarity (99.9%) to the GZ2019 strain, which causes death and severe clinical symptoms in Aratinga solstitialis. Furthermore, when compared to other strains of PaBV-4, the GD2207 strain demonstrated the highest amino acid homology with GZ2019. The phylogenetic analysis demonstrated that the GD2207 strain clustered with various strains found in Japanese, American, and German parrots, indicating a close genetic relationship with PaBV-4, but it revealed a distant relationship with PaBV-5 Cockg5 from America. Notably, the GD2207 was closely associated with the GZ2019 strain from Aratinga solstitialis in China. CONCLUSION: This study presents the preliminary identification of PaBV-4 in Amazona aestiva parrots, emphasizing its importance as the predominant viral genotype linked to parrot infections resulting from trade into China. Through genetic evolution analysis, it was determined that the GD2207 strain of PaBV-4 exhibits the closest genetic relationship with GZ 2019 (Aratinga solstitialis, China), M14 (Ara macao, USA), AG5 (Psittacus erithacus, USA) and 6758 (Ara ararauna, Germany) suggesting a shared ancestry.

Factors Influencing Vertical Transmission of Psittacine Bornavirus in Cockatiels (Nymphicus hollandicus).

The transmission of parrot bornavirus is still not fully understood. Although horizontal transmission through wounds can be one route, vertical transmission is still discussed. PaBV RNA and PaBV antigen were detected in psittacine embryos, but isolation of the virus failed, raising doubts about this route. In this study, cockatiels were infected either as adults (adult group) or during the first 6 days after hatching (juvenile group) and raised until sexual maturity to breed and lay eggs. A total of 92 eggs (adult group: 49, juvenile group: 43) were laid and incubated until day 17. The embryos and yolk samples were examined by RT-PCR for PaBV RNA and by infectivity assay for infectious virus. In the adult group, 14/31 embryos (45.2%) and 20/39 (51%) of the yolk samples demonstrated PaBV RNA in the PCR. Isolation of PaBV was not possible in any embryo of this group, but it was achieved in six yolk samples from one female. Anti-PaBV antibodies were detected in the yolk samples after seroconversion of all female parents. In the juvenile group, 22/29 embryos (74.9%) were positive for PaBV RNA. In 9/21 embryos (42.9%), PaBV isolation was possible. PaBV RNA was detected in 100% and infectious virus in 41% of the yolk samples. Anti-PaBV antibodies were detected in all yolk samples. For the first time, successful vertical transmission of PaBV was proven, but it seems to depend on the age when the parent birds are infected. Therefore, the age of the bird at time of infection may be an important factor in the occurrence of vertical transmission.

Tissue Distribution of Parrot Bornavirus 4 (PaBV-4) in Experimentally Infected Young and Adult Cockatiels (Nymphicus hollandicus).

Proventricular dilatation disease (PDD) caused by parrot bornavirus (PaBV) infection is an often-fatal disease known to infect Psittaciformes. The impact of age at the time of PaBV infection on organ lesions and tissue distribution of virus antigen and RNA remains largely unclear. For this purpose, tissue sections of 11 cockatiels intravenously infected with PaBV-4 as adults or juveniles, respectively, were examined via histology, immunohistochemistry applying a phosphoprotein (P) antibody directed against the bornaviral phosphoprotein and in situ hybridisation to detect viral RNA in tissues. In both groups of adult- and juvenile-infected cockatiels, widespread tissue distribution of bornaviral antigen and RNA as well as histologic inflammatory lesions were demonstrated. The latter appeared more severe in the central nervous system in adults and in the proventriculus of juveniles, respectively. During the study, central nervous symptoms and signs of gastrointestinal affection were only demonstrated in adult birds. Our findings indicate a great role of the age at the time of infection in the development of histopathological lesions and clinical signs, and thus provide a better understanding of the pathogenesis, possible virus transmission routes, and the development of carrier birds posing a risk to psittacine collections.

Canary Bornavirus (Orthobornavirus serini) Infections Are Associated with Clinical Symptoms in Common Canaries (Serinus canaria dom.).

While parrot bornaviruses are accepted as the cause of proventricular dilatation disease (PDD) in psittacine birds, the pathogenic role of bornaviruses in common canaries is still unclear. To answer the question of whether canary bornaviruses (species Orthobornavirus serini) are associated with a PDD-like disease in common canaries (Serinus canaria f. dom.), the clinical data of 201 canary bird patients tested for bornaviruses using RT-PCR assays, were analyzed for the presence of PDD-like gastrointestinal or central nervous system signs and for other viruses (mainly circovirus and polyomavirus), yeasts and trichomonads. Canary bornavirus RNA was detected in the clinical samples of 40 out of 201 canaries (19.9%) coming from 28 of 140 flocks (20%). All nucleotide sequences obtained could unequivocally be determined as canary bornavirus 1, 2, or 3 supporting the current taxonomy of the species Orthobornavirus serini. PDD-like signs were found associated with canary bornavirus detection, and to a lesser extent, with circoviruses detection, but not with the detection of polyomaviruses, yeasts or trichomonads. The data indicate that canary bornaviruses contribute to a PDD-like disease in naturally infected canaries, and suggest a promoting effect of circoviruses for the development of PDD-like signs.

Experimental Infection of Embryonic Cells and Embryonated Eggs of Cockatiels (Nymphicus hollandicus) with Two Parrot Bornavirus Isolates (PaBV-4 and PaBV-2).

Parrot bornavirus (PaBV) might be transmitted vertically. Cockatiel embryonic brain cells and embryonated eggs of cockatiels (ECE) were infected with PaBV-2 and PaBV-4. In embryonic brain cells, PaBV-2 and PaBV-4 showed no differences in viral spread despite the slower growth of PaBV-2 compared with PaBV-4 in CEC-32 cells. ECE were inoculated with PaBV-4 and 13-14 dpi, organs were sampled for RT-PCR, immunohistochemistry/histology, and virus isolation. In 28.1% of the embryos PaBV-4-RNA and in 81.3% PaBV-4-antigen was detected in the brain. Virus isolation failed. Division of organ samples and uneven tissue distribution of the virus limited the results. Therefore, 25 ECE were inoculated with PaBV-4 (group 1) and 15 ECE with PaBV-2 (group 3) in the yolk sac, and 25 ECE were inoculated with PaBV-4 (group 2) and 15 eggs with PaBV-2 (group 4) in the chorioallantoic membrane to use the complete organs from each embryo for each examination method. PaBV-RNA was detected in the brain of 80% of the embryos in groups 1, 2, 3 and in 100% of the embryos in group 4. In 90% of the infected embryos of group 1, and 100% of group 2, 3 and 4, PaBV antigen was detected in the brain. PaBV antigen-positive brain cells were negative for anti-neuronal nuclear protein, anti-glial fibrillary acidic protein, and anti S-100 staining. Virus was not re-isolated. These results demonstrated a specific distribution pattern and spread of PaBV-4 and PaBV-2 in the brain when inoculated in ECE. These findings support a potential for vertical transmission.

Vasculitis Associated with Parrot Bornavirus 4 Infection in a Rose-Crowned Parakeet (Pyrrhurarhodocephala).

A 3-month-old, female rose-crowned parakeet (Pyrrhura rhodocephala) was found dead after a 24-h course of lethargy and passing blood-tinged faeces. Fine white streaks were seen in the pectoral muscles on necropsy. Microscopic examination revealed typical lesions of avian ganglioneuritis and vascular necrosis in the pectoral muscles, myocardium, kidneys, air sacs, adrenal glands, pancreas and thyroid gland. These lesions were characterized by mural fibrinoid necrosis of small and medium-calibre arteries and arterioles, associated with lymphoplasmacytic inflammation, necrosis, atrophy and fibrosis of the surrounding tissues. Parrot bornavirus (PaBV) nucleoprotein was demonstrated by immunohistochemistry in smooth muscle and endothelial cells of many vessels. An avian bornavirus was isolated from kidney tissue and its identity confirmed as PaBV-4 by sequencing and phylogenetic analysis. We postulate that the vascular lesions could have been immune-mediated and that PaBV-4 may have played a role in its pathogenesis.

Avian Bornavirus Research-A Comprehensive Review.

Avian bornaviruses constitute a genetically diverse group of at least 15 viruses belonging to the genus Orthobornavirus within the family Bornaviridae. After the discovery of the first avian bornaviruses in diseased psittacines in 2008, further viruses have been detected in passerines and aquatic birds. Parrot bornaviruses (PaBVs) possess the highest veterinary relevance amongst the avian bornaviruses as the causative agents of proventricular dilatation disease (PDD). PDD is a chronic and often fatal disease that may engulf a broad range of clinical presentations, typically including neurologic signs as well as impaired gastrointestinal motility, leading to proventricular dilatation. It occurs worldwide in captive psittacine populations and threatens private bird collections, zoological gardens and rehabilitation projects of endangered species. In contrast, only little is known about the pathogenic roles of passerine and waterbird bornaviruses. This comprehensive review summarizes the current knowledge on avian bornavirus infections, including their taxonomy, pathogenesis of associated diseases, epidemiology, diagnostic strategies and recent developments on prophylactic and therapeutic countermeasures.

Immunophenotype of the inflammatory response in the central and enteric nervous systems of cockatiels (Nymphicus hollandicus) experimentally infected with parrot bornavirus 2.

Proventricular dilatation disease is a lethal disease of psittacine birds. In this study, we characterized the local cellular immune response in the brain, proventriculus, and small intestine of 27 cockatiels (Nymphicus hollandicus) experimentally infected with parrot bornavirus 2 (PaBV-2). Perivascular cuffs in the brain were composed of CD3+ T-lymphocytes and Iba1+ macrophages/microglia in most cockatiels (n = 26). In the ganglia of the proventriculus, CD3+ T-lymphocytes (n = 17) and Iba1+ macrophages (n = 13) prevailed. The ganglia of the small intestine had a more homogeneous distribution of these leukocytes, including PAX5+ B-lymphocytes (n = 9), CD3+ T-lymphocytes (n = 8), and Iba1+ macrophages (n = 8). Our results indicate that perivascular cuffs in the brain and the inflammatory infiltrate in the proventriculus of PaBV-2-infected cockatiels is predominately composed of T-lymphocytes, while the inflammatory infiltrates in the ganglia of the small intestine are characterized by a mixed infiltrate composed of T-lymphocytes, B-lymphocytes, and macrophages.

A New Multiplex Real-Time RT-PCR for Simultaneous Detection and Differentiation of Avian Bornaviruses.

Avian bornaviruses were first described in 2008 as the causative agents of proventricular dilatation disease (PDD) in parrots and their relatives (Psittaciformes). To date, 15 genetically highly diverse avian bornaviruses covering at least five viral species have been discovered in different bird orders. Currently, the primary diagnostic tool is the detection of viral RNA by conventional or real-time RT-PCR (rRT-PCR). One of the drawbacks of this is the usage of either specific assays, allowing the detection of one particular virus, or of assays with a broad detection spectrum, which, however, do not allow for the simultaneous specification of the detected virus. To facilitate the simultaneous detection and specification of avian bornaviruses, a multiplex real-time RT-PCR assay was developed. Whole-genome sequences of various bornaviruses were aligned. Primers were designed to recognize conserved regions within the overlapping X/P gene and probes were selected to detect virus species-specific regions within the target region. The optimization of the assay resulted in the sensitive and specific detection of bornaviruses of Psittaciformes, Passeriformes, and aquatic birds. Finally, the new rRT-PCR was successfully employed to detect avian bornaviruses in field samples from various avian species. This assay will serve as powerful tool in epidemiological studies and will improve avian bornavirus detection.

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.

Wounds as the Portal of Entrance for Parrot Bornavirus 4 (PaBV-4) and Retrograde Axonal Transport in Experimentally Infected Cockatiels (Nymphicus hollandicus).

In this study, we investigated the natural route of infection of psittacine bornavirus (PaBV), which is the causative agent of proventricular dilatation disease (PDD) in psittacines. We inoculated two infection groups through wounds with a PaBV-4 isolate. In nine cockatiels (Nymphicus hollandicus) we applied a virus suspension with a titer of 103 50% tissue culture infection dose (TCID50) via palatal lesions (Group P, P1-9). In a second group of three cockatiels, we applied a virus suspension with a titer of 104 TCID50 to footpad lesions (Group F, F1-3). In two cockatiels, the control (or "mock") group, we applied a virus-free cell suspension (Group M, M1-2) via palatal lesions. The observation period was 6 mo (Groups P and M) or 7 mo (Group F). We monitored PaBV-4 RNA shedding and seroconversion. At the end of the study, we examined the birds for the presence of inflammatory lesions, PaBV-4 RNA, and antigen in tissues, as well as virus reisolation of brain and crop material. We did not observe any clinical signs typical of PDD during this study. We also did not see seroconversion or PaBV RNA shedding in any bird during the entire investigation period, and virus reisolation was not successful. We only found PaBV-4 RNA in sciatic nerves, footpad tissue, skin, and in one sample from the intestine of Group F. In this group, the histopathology revealed mononuclear infiltrations mainly in skin and footpad tissue; immunohistochemistry showed positive reactions in spinal ganglia and in the spinal cord, and slightly in skin, footpad tissues, and sciatic nerves. In Groups P and M we found no viral antigen or specific inflammations. In summary, only the virus application on the footpad lesion led to detectable PaBV RNA, mononuclear infiltrations, and positive immunohistochemical reactions in tissues of the experimental birds. This could suggest that PaBV spreads via nervous tissue, with skin wounds as the primary entry route.

Update on Avian Bornavirus and Proventricular Dilatation Disease: Diagnostics, Pathology, Prevalence, and Control.

Avian bornavirus (ABV) is a neurotropic virus that can cause gastrointestinal and/or neurologic signs of disease in birds. The disease process is called proventricular dilatation disease (PDD). The characteristic lesions observed in birds include encephalitis and gross dilatation of the proventriculus. ABV is widely distributed in captive and wild bird populations. Most birds infected do not show clinical signs of disease. This article is an update of the Veterinary Clinics of North America article from 2013: Avian Bornavirus and Proventricular Dilatation Disease: Diagnostics, Pathology, Prevalence, and Control.

Cardiac Lesions of Natural and Experimental Infection by Parrot Bornaviruses.

Lymphoplasmacytic inflammation associated with bornavirus N protein occurs in the epicardial ganglia, myocardium and endocardium of birds diagnosed with proventricular dilatation disease (PDD). These pathological findings suggest that sudden death in psittacine birds might stem from cardiac compromise due to parrot bornavirus (PaBV) infection. Therefore, we investigated cardiac lesions in cases of PDD, searching databases from 1988 to 2019, and reviewed three experimental studies of PaBV infection. Fifty cases of PDD in birds infected naturally with PaBV and 27 cases of PDD in birds infected experimentally with PaBV (all having descriptions of inflammatory cardiac lesions) were reviewed. For each case, five regions of the heart were evaluated by light microscopy and immunohistochemistry (IHC). These regions were the epicardial ganglia/nerves, the endocardium, the myocardium, the Purkinje fibres and the great vessels. Sudden death was documented in 17/50 naturally infected cases, while 23/50 had digestive signs, and only 12/50 had neurological signs. Grossly, only five naturally-infected and five experimentally-infected cases had cardiomegaly or hydropericardium. Epicardial ganglioneuritis was the most consistent microscopical finding in natural (46/50) and experimental cases (26/27), followed by myocarditis (34/50) for naturally-infected and endocarditis for experimentally-infected birds (6/27). PaBV-2 antigen was detected most frequently by IHC in the epicardial ganglia (54/77) compared with the other tissues. This retrospective study demonstrates the presence of PaBV protein and inflammation in the heart of birds infected with PaBV and suggests a link between PaBV and cardiac disease and sudden death in psittacine birds.

Monitoring of free-ranging and captive Psittacula populations in Western Europe for avian bornaviruses, circoviruses and polyomaviruses.

Avian pathogens such as bornaviruses, circoviruses and polyomaviruses are widely distributed in captive collections of psittacine birds worldwide and can cause fatal diseases. In contrast, only little is known about their presence in free-ranging psittacines and their impact on these populations. Rose-ringed parakeets (Psittacula krameri) and Alexandrine parakeets (Psittacula eupatria) are non-native to Europe, but have established stable populations in parts of Western Europe. From 2012-2017, we surveyed free-ranging populations in Germany and France as well as captive Psittacula individuals from Germany and Spain for avian bornavirus, circovirus and polyomavirus infections. Samples from two out of 469 tested free-ranging birds (0.4%; 95% confidence interval [CI-95]: 0.1-1.5%) were positive for beak and feather disease virus (BeFDV), whereas avian bornaviruses and polyomaviruses were not detected in the free-ranging populations. In contrast, avian bornaviruses and polyomaviruses, but not circoviruses were detected in captive populations. Parrot bornavirus 4 (PaBV-4) infection was detected by RT-PCR in four out of 210 captive parakeets (1.9%; CI-95: 0.7-4.8%) from four different holdings in Germany and Spain and confirmed by detection of bornavirus-reactive antibodies in two of these birds. Three out of 160 tested birds (1.9%; CI-95: 0.5-5.4%) possessed serum antibodies directed against budgerigar fledgling disease virus (BuFDV). PaBV-4 and BuFDV were also detected in several psittacines of a mixed holding in Germany, which had been in contact with free-ranging parakeets. Our results demonstrate that Psittacula parakeets are susceptible to common psittacine pathogens and their populations in Western Europe are exposed to these viruses. Nevertheless, the prevalence of avian bornaviruses, circoviruses and polyomaviruses in those populations is very low.RESEARCH HIGHLIGHTS Psittacula parakeets are susceptible to bornavirus, circovirus and polyomavirus infection.Introduced Psittacula populations in Europe have been exposed to these viruses.Nevertheless, they may be absent or present at only low levels in free-ranging Psittacula populations.Free-ranging populations in Europe pose a minor threat of transmitting these viruses to captive Psittaciformes.