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.

The modulating effect of food composition on the immune system in growing ring-necked pheasants (Phasianus colchicus).

The decline in the population of ring-necked pheasants (Phasianus colchicus) in northwestern Germany since 2007 raises questions about the underlying causes. We therefore studied the growth and immune status of ring-necked pheasant chicks dependent on different feed composition. Here, 490 ring-necked pheasant chicks were raised in five groups up to nine weeks. While control groups C1 and C2 received sufficient crude protein (28%) and energy (12.5 MJ/Kg feed) according to current standards, group C2 was treated with cyclosporine eight hours prior to phythemagglutination (PHA) testing, serving as a positive immune suppressed control. Group V1 was fed with reduced protein (20%) but optimal energy content (12.5 MJ/Kg feed), group V2 was fed with sufficient protein (28%) and reduced energy content (10 MJ/kg feed) whereas group V3 was fed reduced crude protein (20%) and reduced energy content (10MJ/kg feed). On all chicks, health status was checked each week, and 20 birds of each group were weighed randomly per week. PHA-testing was performed on 12 birds of each group to study the in vivo non-specific activation of lymphocytes at week 2, 4, 6, 7, 8 and 9. In addition, hemolysis-hemagglutination-assay (HHA) was performed on each of the PHA-tested chicks, which were subsequently euthanized and dissected. Histopathologic examinations of 5 birds that were randomly chosen were performed. The PHA-test results demonstrate significant differences between control (C1, C2) and experimental groups (V1-V3) in several developmental stages. According to the HHA results, weekly testing detected a significant increase of titres per week in all groups without significant differences. Here, only hemagglutination and no lysis of samples was observed. It seems appropriate to conclude that during their first weeks of life, protein content is of higher importance in ring-necked pheasant chicks than energy intake. In particular T-cell response is significantly reduced, which indicate a weaker immune system resulting in a higher risk for clinical diseases. Therefore, we assume that protein i.e. insect availability is a highly important co-factor in the free-ranging population dynamics, and is linked to declines of the northwestern German population.

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.

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.