Psittacine beak and feather disease (PBFD) is a readily recognisable dermatologic
condition in wild and captive psittacines worldwide. It is caused by Beak and feather
disease virus (BFDV) which is classified in the family Circoviridae and the genus
Circovirus. BFDV has a circular ss-DNA genome consisting of seven open reading
frames (ORFs), three being conserved in all BFDV isolates, ORF 1 which encodes
the Rep protein, ORF 2 which encodes the coat or capsid protein (CP) and ORF 5
which encodes a protein whose function is as yet unknown. General symptoms of the
disease include the symmetrical loss of feathers, feather abnormalities, beak and
claw deformities, weight loss, anorexia and immunosuppression. The inability to grow
BFDV in tissue culture or in embryonated eggs has hindered the routine diagnosis of
PBFD affected birds and the development of reliable diagnostic tests and an effective
vaccination program.
PBFD is widespread in South Africa, leading to a loss of at least 10% of psittacine
breeding stocks annually. The disease is also a major threat to the already
endangered Cape Parrot (Poicephalus robustus) and the black-cheeked lovebird
(Agapornis nigrigenis) and it is only a matter of time before we may see the extinction
of these and other parrot species due to the lack of a preventative vaccine. The
economical and natural implications of the attack by PBFD led to the aims of the
present study which were to develop a potential DNA vaccine candidate, develop an
expression system for production of recombinant CP as antigenic protein and
establish an enzyme linked immunosorbent assay for the detection of BFDV-specific
antibodies in parrots.
The entire CP gene which has been suggested to encode for the epitopic protein of
the virus was amplified by polymerase chain reaction (PCR) and ligated into a
bacterial vector, pBAD/His B or a yeast vector, pKOV136 for expression of
recombinant CP in Escherichia coli or Yarrowia lipolytica, respectively. Alternatively,
CP gene PCR products were ligated into the mammalian expression vector
pcDNAâ¢3.1D/V5-His-TOPO® which was the vector of choice for DNA vaccine design
and used to transiently transfect Chinese hamster ovary cells. Subsequently, the
candidate DNA vaccine was used in a basic vaccine trial where budgerigars
(Melopsittacus undulatus) were vaccinated either with the DNA vaccine candidate or
a sub-unit vaccine consisting of purified recombinant CP. Expression of recombinant CP was monitored using polyacrylamide gel electrophoresis (PAGE),
chemiluminescent and colorimetric detection on Western blots and ELISAs.
While expression of the recombinant CP was unsuccessful in the yeast system using
pKOV136, expression of recombinant CP was achieved in E. coli cells using the
pBAD vector. Recombinant CP was partially purified and applied in both indirect and
indirect competitive ELISAs as coating antigen for the detection of BFDV specific
antibodies. Using the established ELISAs, BFDV specific antibodies could be
detected in naturally infected parrots as well as in budgerigars vaccinated with the
DNA vaccine and sub-unit vaccine. Comparable results were obtained when nonpurified
recombinant CP was applied in the ELISAs in lieu of partially purified
recombinant CP.
Vaccinated budgerigars formed BFDV specific antibodies in response to the DNA
vaccine and sub-unit vaccine that were detected using the indirect competitive ELISA
established in the study. The antibody responses to the sub-unit vaccine were higher
than those in response to vaccination with the DNA vaccine candidate. Although the
indirect competitive ELISA could not provide an indication of whether these antibody
responses are protective, the results obtained during the trial are a preliminary
indication that both the DNA vaccine and sub-unit vaccine may be functional in
parrots and safe to use as no adverse reactions were observed.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-04082009-151418 |
| Date | 08 April 2009 |
| Creators | Kondiah, Kulsum |
| Contributors | Prof J Albertyn, Prof RR Bragg |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-04082009-151418/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
Page generated in 0.0146 seconds