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

Abstract

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.

Keywords: avian; central nervous system; nerval disease; proventricular dilatation disease; psittacines; species conservation; vertical transmission.

Figure 1

Uninfected embryonic cockatiel brain cells, including a large pleomorphic cell with two nuclei (white arrow) alongside smaller cells with multiple nuclei (yellow arrow).

Figure 2

Indirect immunofluorescence assay: PaBV-4-infected embryonic cockatiel brain cells with granules in the nuclei (white arrow).

Figure 3

Parrot bornavirus 4 (PaBV-4) antigen detection in the brain of trial B embryos. (A) Cerebellum with PaBV antigen-positive brain cells, DAB staining in the arbor vitae (embryo DAB_PABV-2_01, 100× total magnification, size bar 200 µm). (B) Cerebrum with ventricle, positive brain cells by DAB staining at the base of ependymal cells (Embryo DAB_PABV-4_DO01, 200× total magnification, size bar 100 µm). (C) Cerebrum without ventricle with only a few positive cells visualized by DAB staining (embryo DAB_PABV-2_01, 100× total magnification, size bar 200 µm).

Figure 3

Parrot bornavirus 4 (PaBV-4) antigen detection in the brain of trial B embryos. (A) Cerebellum with PaBV antigen-positive brain cells, DAB staining in the arbor vitae (embryo DAB_PABV-2_01, 100× total magnification, size bar 200 µm). (B) Cerebrum with ventricle, positive brain cells by DAB staining at the base of ependymal cells (Embryo DAB_PABV-4_DO01, 200× total magnification, size bar 100 µm). (C) Cerebrum without ventricle with only a few positive cells visualized by DAB staining (embryo DAB_PABV-2_01, 100× total magnification, size bar 200 µm).

Figure 4

Parrot bornavirus (PaBV) antigen distribution in the brain of trial B group 1 (PaBV-4 inoculation in the yolk sac). The y-axis shows the number of PaBV antigen-positive cells counted in one slide (200× total magnification). PaBV antigen was mainly found in the cerebellum and cerebrum with ventricles, and much less frequently in the cerebrum without ventricles. The x-axis shows each individual embryo (n = 10, laboratory number).

Figure 5

Parrot bornavirus (PaBV) antigen distribution in the brain of trial B group 2 (PaBV-4 inoculation in the chorioallantoic membrane). The y-axis shows the number of PaBV antigen-positive cells counted in one slide (200× total magnification). The PaBV antigen distribution similar to trial B group 1. The x-axis shows each individual embryo (n = 10, laboratory number).

Figure 6

Parrot bornavirus (PaBV) antigen distribution in the brain trial B group 3 (PaBV-2 inoculation in the yolk sac). The y-axis shows the number of PaBV antigen-positive cells counted in one slide (200× total magnification). The PaBV antigen distribution is similar to trial B groups 1 and 2. There was no significant difference between the cerebrum with and without ventricles. The x-axis shows each individual embryo (n = 5, laboratory number).

Figure 7

Parrot bornavirus (PaBV) antigen distribution in the brain of trial B group 4 (PaBV-2 inoculation in the chorioallantoic membrane). The y-axis shows the number of PaBV antigen-positive cells counted in one slide (200× total magnification). The PaBV antigen distribution is similar to trial B group 3. There was no significant difference between the cerebellum and the cerebrum without ventricles and between the cerebellum and the cerebrum with ventricles. There was a significant difference between the cerebrum with and without ventricles. The x-axis shows each individual embryo (n = 5, laboratory number).