Establishment of serological and molecular techiques to investigate diversity of psittacine beak and feather disease virus in different psittacine birds in South Africa
Psittacine beak and feather disease (PBFD) is a readily recognizable disease of wild and captive psittacines in Australia but it is also a problem worldwide wherever captive species are bred. The disease caused by beak and feather disease virus (BFDV) is characterized by the progressive development of feather dystrophy and loss. Although the occurrence of PBFD in South Africa has been reported only recently, it is already rampant and threatens the extinction of the endangered Cape parrot and black-cheeked lovebird. Currently no vaccine for PBFD is commercially available but the loss of approximately 10-20% of breeding stocks of psittacines annually in South Africa alone is enough to realise the importance of one. Genetic and antigenic differences in BFDV are significant aspects for production of a vaccine but the lack of a culture system for BFDV has limited studies into its genetics, antigenicity and pathogenicity. The objective of the study thus became to establish techniques that could be used to investigate genetic and antigenic differences that may be present in BFDV in South African psittacines. Molecular investigations involved the testing dried blood samples for BFDV nucleic acid using polymerase chain reaction (PCR). A region within the Rep gene was amplified and digested with HaeIII to yield restriction length fragment polymorphisms (RFLPs). Six RFLPs were identified, cloned, sequenced (UFS 1- 6) and phylogenetically analysed. BFDV was purified from body organs of PBFD- affected birds by cesium chloride density gradient centrifugation and fractions tested by PCR and haemagglutination (HA) assays. BFDV-specific antibodies were raised in two rabbits by inoculation with purified BFDV in Freund’s incomplete adjuvant and tested by haemagglutination inhibition (HI) assays and an enzyme-linked immunosorbent assay (ELISA). HA and HI assays were attempted using erythrocytes from African grey parrots and Brown-headed parrots. HI assays were also used to test parrot sera for the presence of antibodies to BFDV. UFS 1-6 were closely related to known BFDV isolates and UFS 1 may represent a unique genotype in South Africa as it was separated from its closely related isolates by a 90% bootstrap value. UFS 3, 4 and 5 isolated from budgerigars belong to the budgerigar lineage as they clustered with isolate BG3-NZ. Together with three previously identified genotypes, UFS 1 indicates the introduction of BFDV into southern Africa on four separate occasions. Purification of BFDV from organs was successful but yielded low titres possibly because low quantities of virus were present in the organs or because little virus was circulating in the bird upon its death. PCR and HA assays confirmed the presence of BFDV in fractions; the two results correlated well. HA and HI assays were successfully established using erythrocytes from African grey parrots and Brown-headed parrots. Antibodies to BFDV were successfully detected in sera of three parrots by the HI assay. However, the assay could not detect non-psittacine raised antibodies (rabbit-raised) due to non-specific reactions. BFDV-specific antibodies were successfully raised in rabbits and were verified by the use of an ELISA. The high level of genetic diversity observed in the study compels further investigation into the genetics of BFDV as such levels of diversity may become a limiting factor in the applicability of PCR as a diagnostic test. The entire diversity of BFDV has not been studied and future work may lead to the identification of more BFDV strains, an important factor in vaccine development. The rabbit- raised antibodies together with the HA and HI assays and ELISA can be used to study the antigenic differences that may be present in known BFDV isolates that may also lead to the identification of more strains of the virus.
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