Ursula Heffels-Redmann

Pathogenesis of Avian Bornavirus in Experimentally Infected Cockatiels

Inoculation induced persistent infection, clinical signs, and seroconversion.

Indirect Immunofluorescence Assay for Intra Vitam Diagnosis of Avian Bornavirus Infection in Psittacine Birds

ABSTRACT Different avian bornavirus (ABV) genotypes have recently been detected in psittacine birds with proventricular dilatation disease (PDD), an inflammatory fatal central and peripheral nervous system disorder. An indirect immunofluorescence assay (IIFA) for intra vitam demonstration of ABV-specific serum antibodies was established since reverse transcription-PCR (RT-PCR) assays may not detect all ABV variants.

Structural and biological characteristics of reoviruses isolated from Muscovy ducks (Cairina moschata)

Two Muscovy duck reoviruses, strains y1/79 and 1625/87, were investigated with regard to their genome organization, polypeptide pattern, serotype specificity, and pathogenicity. Electrophoretic analysis of the genome revealed the migration pattern of avian reoviruses. In spite of general conformity, great polymorphism was detected in the electrophoretic mobility of individual genome segments of the two strains sharing only three segments of identical size (L1, M2, M3). Only one segment (M3) migrated into the same position as the corresponding segment of prototype chicken reovirus S1133. The basic electrophoretic mobility pattern of the immunoprecipitated polypeptides, eight structural and two non-structural, closely resembled that of the chicken reovirus. However, considerable strain-specific variation was also seen at the protein level, with the sigma(c) polypeptides exhibiting the most obvious migration differences. Based on the results of cross-neutralization assays the two Muscovy duck reovirus strains were grouped into one serotype, with no cross-reactivity to the chicken serotype S1133. In experimental infections, despite virus replication proved by faecal virus excretion and antibody response, only strain y1/79 was pathogenic for 16-day-old Muscovy ducklings, thus making strain 1625/87 a possible candidate as vaccine strain.

Occurrence of avian bornavirus infection in captive psittacines in various European countries and its association with proventricular dilatation disease.

A total of 1442 live birds and 73 dead birds out of 215 bird collections in Spain, Germany, Italy, the UK and Denmark were tested for avian bornavirus (ABV) infection by four different methods. The majority of the birds were psittacines belonging to 54 different genera of the order Psittaciformes. In total, 22.8% of the birds reacted positive for ABV in at least one of the tests. Combined testing of swabs from the crop and cloaca, and serum for the diagnosis of ABV infection in live birds revealed that virus shedding and antibody production coincided in only one-fifth of the positive birds so that the examination of these three samples is recommended for reliable ABV diagnosis. By statistical analysis of this large number of samples, the ABV infection proved to be highly significant (P <0.001) associated with histopathologically confirmed proventricular dilatation disease (PDD) in dead birds as well as with clinically assumed PDD in live birds. However, ABV infection was also detected in psittacines without pathological lesions or clinical signs of PDD. Twelve non-psittacine birds belonging to the genera Aburria, Ciconia, Geopelia, Leucopsar and Pavo were tested negative for ABV infection. Within the order of Psittaciformes, birds belonging to 33 different genera reacted positive for ABV. In 16 of these psittacine genera, the ABV infection was demonstrated for the first time. The present study emphasizes the widespread occurrence of clinically variable ABV infections in Europe by analysing a large number of specimens from a broad range of bird species in several assays.

Follow-Up Investigations on Different Courses of Natural Avian Bornavirus Infections in Psittacines

SUMMARY. To study the course of natural avian bornavirus (ABV) infection, 63 psittacines of three bird collections where ABV had been demonstrated were investigated over a period of 1 yr. The psittacines were clinically observed and swabs of crop and cloaca as well as serum samples were collected three separate times at intervals of 2–6 mo. According to the results of detection of ABV RNA by reverse transcriptase polymerase chain reaction (RT-PCR) and of anti-ABV antibodies by indirect immunofluorescence assay (IIFA), 43 of the birds were found to be infected with ABV. Based on variations in virus shedding and antibody production in combination with the occurrence of proventricular dilatation disease (PDD) –related clinical signs, pathological findings, and lethal outcome, four different groups of infected psittacines and a fifth group of noninfected psittacines were identified. Group 1 comprised six birds with various courses of ABV infection and forms of clinical PDD. Groups 2–4 included all birds with subclinical ABV infections: Group 2 contained 13 birds that were consistently (subgroup A, 6 birds) or inconsistently (subgroup B, 7 birds) ABV positive by PCR and serology; group 3 was composed of 13 psittacines exhibiting only anti-ABV antibodies; and 8 birds that had positive ABV RNA detection in crop and cloaca, but did not develop anti-ABV specific antibodies, were classified in group 4. Twenty-three out of the 63 psittacines remained free of detectable ABV RNA or anti-ABV antibodies over the whole observation period (group 5). Based on the results, it seems that birds with high ABV RNA load in crop and cloaca combined with high anti-ABV antibodies have a high risk of the development of PDD, indicating that the humoral antibodies do not protect against the disease. The meaning of the detection of ABV RNA and antibodies at a low and inconsistent level for the single bird as well as for the epidemiology of the ABV infection remained unclear in this field study and needs to be further investigated.

Serologic Testing for Avian Influenza Viruses in Wild Birds: Comparison of Two Commercial Competition Enzyme-Linked Immunosorbent Assays

Abstract Serologic testing of wild birds for avian influenza virus (AIV) surveillance poses problems due to species differences and nonspecific inhibitors that may be present in sera of wild birds. Recently available competitive enzyme-linked immunosorbent assay (cELISA) kits offer a new species-independent approach. In this study we compare two commercial competitive cELISAs, using a total of 184 serum and plasma samples from 23 species of wild birds belonging to 10 orders. Thirteen samples were from experimentally high pathogenicity AI and low pathogenicity AI infected red-legged partridges (Alectoris rufa), 77 samples were from a flock of sentinel hybrid ducks confirmed infected by AI by real-time PCR, and 94 samples were from wild birds admitted to a rehabilitation center. Both ELISAs detected AI antibodies in the experimentally infected partridges, whereas hemagglutination inhibition (HI) was negative. Concordance in results between the two ELISAs was 51.5%. When specific subtype-H5/H7 HI-positive samples were considered for comparison, ELISA 1 appeared to perform better on ducks, whereas ELISA 2 appeared to perform better in other wild bird species. Overall, 68.2% of H5/H7 positive samples tested positive by ELISA 1 and 36% by ELISA 2. Both ELISAs detected AIV-antibody–positive samples negative by specific HI against 9 of the 16 existing hemagglutinin (HA) subtypes. Presumably this reflects either higher sensitivity of cELISA when compared to HI, presence of antibodies against HA subtypes not tested, or unspecific reactions. Performance of ELISA 1 on ducks appears to be comparable to in-house cELISA previously used by other authors in wild birds, but requires a relatively large sample volume. Alternatively, although ELISA 2 required a smaller sample volume, it was less effective at identifying HI-positive samples. The results reflect the necessity of validation of cELISA tests for individual species or at least families, as required by the OIE.

Vertical Transmission of Avian Bornavirus in Psittacines

To the Editor: Proventricular dilatation disease (PDD) is a fatal disease in psittacines that jeopardizes critical species conservation projects, such as that involving the Spix’s macaw (Cyanopsitta spixii), the world’s most endangered bird species (1). The disease is characterized by lymphoplasmacytic infiltrations in the enteric and central nervous systems (2). Consequently, gastrointestinal and neurologic disorders are the major clinical manifestations. Only recently has the cause of the disease been identified by characterization of a newly discovered member of the family Bornaviridae, the avian bornavirus (ABV), which has been detected in affected psittacines (3,4). The relationship of an infection with ABV and the occurrence of PDD has been described in natural cases (5,6) and in experimental trials (7,8). However, birds that are infected with ABV but that are clinically healthy have also been recognized (6). Infected birds can shed viral RNA intermittently (9), and not all infected birds seroconvert (5). For psittacine flock management, control of an AΒV infection is critical, e.g., by repeated testing of breeding stock and removal of ABV-positive birds (2,5). However, in breeding projects of rare species, every individual is genetically important and cannot be lost. Therefore, pairing infected, but clinically healthy, birds separately from birds that test negative for the virus might represent an option. For this possibility to be viable, whether vertical transmission of ABV can take place must be further clarified. A study investigating the distribution of ABV in tissues of PDD-positive birds has demonstrated ABV antigen in follicular cells, which may point toward vertical transmission (9). To investigate vertical transmission of ABV, we examined 30 dead-in-shell embryos of various psittacine species that originated from ABV-infected flocks with a history of PDD. First, the eggshell was disinfected and opened at the blunt end by using sterile equipment. The brain and proventriculus of each embryo were analyzed for the presence of ABV RNA by using 2 different real-time reverse transcription PCRs, as described by Honkavuori (4), with the primer pair 1034–1322 and, in case of a negative result, the additional primer set 1367. Sampling, RNA extraction, and PCR were repeated by using the same methods to exclude possible cross-contamination of samples. Afterwards, the complete embryo was placed in 10% buffered formalin, and histopathologic examination and immunohistochemical (IHC) testing were carried out (10) with antibodies directed against the viral phosphoprotein and X protein. If ABV RNA or ABV antigen was demonstrated, crop and cloacal swab specimens and serum of the parents of the positive embryo were immediately taken and used either for ABV RNA detection with the above described PCR or for the detection of specific ABV antibodies by indirect immunofluorescence assay (10). This procedure was chosen because earlier sampling of the parents might have caused breeding interruption, and which eggs of which parents would be available for investigation was not clear. In 2 of the 30 embryos investigated, ABV RNA was detected by using the 1034 PCR. The repeated procedure provided the same results in the same embryos. One embryo was a Major Mitchell cockatoo (Cacatua leadbeateri) (cycle threshold 31.41) and the other a red-crowned Amazon (Amazona viridigenalis) (cycle threshold 35.1). None of the investigated embryos demonstrated histopathologic lesions typical of an ABVinfection. IHC testing did not show any positive results. However, in the ABV-positive Amazon embryo, an equivocal result was obtained. The swab specimens of both parents of the Major Mitchell cockatoo tested positive for ABV RNA, but serum did not demonstrate specific ABV antibodies. The crop swab specimen of the female red crowned Amazon was positive for ABV RNA, but serum was negative for ABV antibodies; the male bird tested negative by PCR but demonstrated an ABV-specific antibody titer of 80. These results highlight the potential risk for vertical transmission of ABV and the conclusion that ABV-infected parents can most likely produce infected offspring. However, test results were positive for only 2 of 30 eggs. Because potentially dead-in-shell embryos are usually further incubated by the breeder to ensure embryonic death, the possibility cannot be excluded that ABV RNA was already degraded in some cases, thus causing false-negative results. On the other hand, these eggs might have originated from ABV-negative parents. The quality of the samples might also have caused the questionable IHC results. In PDD-affected, ABV-positive flocks >30% of the birds could be infected (5,6) and the virus is shed intermittently (9). Therefore, pairing ABV-positive birds, incubating their eggs artificially, and raising the chicks separately until they show negative test results, might be an option for breeding projects. However, when vertical transmission occurs (and, if so, its incidence) is unknown. Whether ABV infection of the embryos was the cause of death remains unclear. Even if typical lesions were not detected, the poor quality of the material might have hidden such lesions. However, embryonic infection that does not result in embryonic death is the basic requirement for successful vertical transmission. These preliminary results warrant further studies investigating the possibility of vertical transmission by ABV-infected pairs, especially to minimize the risk for such transmission to endangered species with restricted breeding opportunities.

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

ABSTRACT 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.

Avian bornavirus in free-ranging psittacine birds, Brazil.

Avian bornavirus (ABV) has been identified as the cause of proventricular dilatation disease in birds, but the virus is also found in healthy birds. Most studies of ABV have focused on captive birds. We investigated 86 free-ranging psittacine birds in Brazil and found evidence for natural, long-term ABV infection.

Investigation of Different Infection Routes of Parrot Bornavirus in Cockatiels

SUMMARY The aim of this study was to determine the natural infection route of parrot bornavirus (PaBV), the causative agent of proventricular dilatation disease (PDD) in psittacines. For this purpose, nine cockatiels (Nymphicus hollandicus) were inoculated orally, and nine cockatiels were inoculated intranasally, with a PaBV-4 isolate. To compare the results of the trials, the same isolate and the same experimental design were used as in a previous study where infection was successful by intravenous as well as intracerebral inoculation. After inoculation, the birds were observed for a period of 6 mo and tested for PaBV RNA shedding, virus replication, presence of inflammatory lesions, and PaBV-4 antigen in tissues, as well as specific antibody production. In contrast to the previous study involving intravenous and intracerebral infections, clinical signs typical for PDD were not observed in this study. Additionally, anti-PaBV antibodies and infectious virus were not detected in any investigated bird during the study. Parrot bornavirus RNA was detected in only four birds early after infection (1–34 days postinfection). Furthermore, histopathologic examination did not reveal lesions typical for PDD, and PaBV antigen was not detected in any organ investigated by immunohistochemistry. In summary, oral or nasal inoculation did not lead to a valid infection with PaBV in these cockatiels. Therefore it seems to be questionable that the formerly proposed fecal-oral transmission is the natural route of infection in immunocompetent adult or subadult cockatiels.