Bragg, R.R.Hattingh, Albertha René2015-08-192015-08-192009-01http://hdl.handle.net/11660/944English: Psittacine beak and feather disease (PBFD) is a dermatological condition that affects both captive and wild psittacine birds worldwide. In Southern Africa, 10 – 20% of breeding stocks are lost due to the disease each year. PBFD threatens the survival of the indigenous endangered Cape parrot (Poicephalus robustus) as well as the black-cheeked lovebird (Agapornis nigrigenis). The disease is characterized by roughly symmetrical feather loss, feather abnormalities, anorexia and immunosuppression. In advanced cases of the disease beak and claw deformities are present. The causative agent of PBFD is Beak and feather disease virus (BFDV), a circovirus belonging to a diverse group of circoviruses within the family Circoviridae. BFDV has a circular single stranded DNA genome consisting of seven open reading frames (ORFs); three of these ORFs are conserved amongst all isolates of BFDV. ORF 1 encodes the Rep protein, ORF 2 the coat protein (CP) which is also the epitopic protein of the virus and ORF 5, whose function remains unclear. BFDV cannot be cultivated in tissue/cell culture or in embryonated eggs. The inability to cultivate the virus has hampered the development of diagnostic tests and a vaccine as preventative measure against the disease. BFDV is a genetically diverse virus. Researchers have demonstrated that there are at least eight different lineages of BFDV, where Southern African isolates group into three unique genotypes. Many studies have been performed which indicated the diversity of BFDV, but so far no studies have been done which link this genetic diversity to the possibility of the existence of more than one strain of BFDV. This led to the aims of the present study which were to investigate antigenicity of BFDV isolates belonging to different genotypes and then the subsequent bacterial expression of six isolates of BFDV that were genetically different. The entire CP genes of six isolates were amplified with polymerase chain reaction (PCR) and subsequently sequenced. Phylogenetic analysis of sequence data showed that the isolates from this study grouped into lineage one as was described by Heath and co-workers (2004). Amino acid sequences from the isolates from each lineage was applied in an in silico prediction algorithm in order to establish the possibility of more than one strain of BFDV. The predictions indicated that isolates from Australia and South Africa had the same antigenic profile. However, isolate BCL1-ZAM and LK-VIC each produced their own antigenic profile. This indicated the distinct possibility that there is more than one strain of BFDV and that at least one antigenic determinant was situated at the N-terminus of the CP. However, these results have to be confirmed by conducting in vitro studies. Attempts were made to express the full length CP genes of six isolates in BL21(DE3) Escherichia coli with the pET-28b(+) vector. Neither polyacrylamide gel electrophoresis (PAGE) nor Western blotting indicated the presence of recombinantly expressed protein in any of the studies conducted. The codon adaptation index (CAI) for BFDV was calculated to be 0.250, which indicated that the CP had a 25% possibility of being expressed in E. coli due to codon incompatibility. From this study it can be suggested that before attempting expression of any gene in E. coli the CAI should be calculated. It was also concluded from this work that no further attempts to express the CP gene in E. coli should be conducted.Afrikaans: Psittacine beak and feather disease (PBFD) is „n dermatologiese toestand wat beide gevange en wilde papagaaie wêrelwyd affekteer. In Suidelike Afrika is daar „n verlies van 10 – 20% van die broeivoëls elke jaar. PBFD bedreig die oorlewing van die alreeds bedreigde inheemse Kaapse papagaai (Poicephalus robustus) sowel as die black-cheeked lovebird (Agapornis nigrigenis). PBFD word gekarakteriseer deur die simmetriese verlies van vere, veerabnormaliteite, anorexia en immuunonderdrukking. In gevorde gevalle van die siekte vertoon voëls bek en klou abnormaliteite. PBFD word veroorsaak deur die Beak and feather disease virus (BFDV), „n circovirus wat behoort aan „n diverse groep van circovirusse geklassifiseer in die familie Circoviridae. BFDV het „n sirkulêre enkelstring DNA genoom wat bestaan uit sewe oopleesrame (OLRe); drie van die OLRe is gekonserveerd in alle BFDV isolate. OLR 1 kodeer vir die Rep proteïen, OLR 2 vir die kapsied proteïen (KP) wat ook die epitopiese proteïen van die virus is en OLR 5 kodeer vir „n proteïen waarvan die funksie onbekend is. BFDV kan nie in weefselkulture of geëmbrioneerde eiers gekweek word nie. Die onvermoë om die virus te kultiveer vermoeilik die ontwikkeling van diagnostiese toetse asook die ontwikkeling van „n entmiddel as voorkoming teen die siekte. BFDV is „n geneties diverse virus. Navorsers het gedemonstreer dat daar ten minste agt verskillende genotipes van BFDV is. Suid Afrikaanse isolate groepeer in drie unieke genotipes. Veelvuldige studies is gedoen wat die diversiteit van BFDV aandui, maar tot dusver is geen studies gedoen wat hierdie genetiese diversiteit koppel aan die moontlikheid van die bestaan van meer as een infektiewe stam van BFDV. Hierdie het gelei tot die doel van die huidige studie wat ingesluit het navorsing in die antigenisiteit 84 van BFDV isolate wat behoort aan verskillende genotipes asook die bakteriese uitdrukking van ses isolate van BFDV wat geneties verskil het. Die volledige KP geen van ses BFDV isolate is geamplifiseer deur polimerase ketting reaksie (PKR) en daarna is die basis paar opeenvolging van die produkte bepaal. Filogenietiese analise van die basis paar opeenvolging data het gewys dat die isolate in die studie gebruik gegroepeer het in die eerste genotipe soos beskryf deur Heath en mede-werkers (2004). Aminosuur opeenvolgings van die isolate is gebruik in „n in silico voorspellings algoritme om te bepaal of daar meer as een stam van BFDV is. Die voorspellings het aangedui dat isolate vanaf Australia en Suid Afrika dieselfde antigeniese profiele gehad het. Die isolate BCL1-ZAM en LK-VIC het elkeen hul eie antigeniese profiel gehad. Die resultate het gedui op die moontlikheid dat daar meer as een infektiewe stam van BFDV is en dat ten minste een antigeniese determinant aan die N-terminus van die KP geleë is. Hierdie bevindings moet bevestig word deur in vitro studies uit te voer. Daar is gepoog om die vollengte KP geen van ses isolate uit te druk in BL21(DE3) Escherichia coli met die pET-28b(+) vektor. Nie poli-akrielamied gel elektroforese (PAGE) of Western blotting het aangedui dat rekombinante proteïen tydens die eksperimente uitgedruk was nie. Die kodon aanpassings indeks (KAI) vir BFDV was bereken om 0.250 te wees, wat aangedui het dat die KP „n 25% kans gehad het om in E. coli uitgedruk te word as gevolg van kodon onversoenbaarheid. Vanuit hierdie studie kan dit voorgestel word dat die CAI bereken moet word voordat uitdrukking van enige geen in E. coli uitgevoer word. Vanaf hierdie studie kan ook bepaal word dat verdere studies om die KP in E. coli uit te druk gestaak moet word.enThesis (M.Sc. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2009ParrotsVirus diseasesGenetic aspectsPsittacine beak and feather disease (PBFD)Beak and feather disease virus (BFDV)SilicoStrainsBacterial expressionCodon adaptation index (CAI)DissertationUniversity of the Free State