Albertyn_29-34_web.qxd INTRODUCTION Psittacine beak and feather disease (PBFD) is a viral disease that was first discovered in 1975 in cockatoos in Australia. It is caused by the psittacine beak and feather disease virus (PBFDV) which is a small (14–18 nm diameter), icosahedral, non- enveloped virus with a circular single-stranded DNA (ssDNA) genome that has been characterised and placed in the family Circoviridae (Raidal, Firth & Cross 1993; Studdert 1993; Pass, Plant & Sexton 1994; Sexton, Penhale, Plant & Pass 1994; Ramis, Latimer, Gibert & Campagnoli 1998; Ypelaar, Bas- sami, Wilcox & Raidal 1999; Todd 2000). The disease occurs predominantly in sulphur-crest- ed cockatoos (Cacatua galerita) but also in budger- igars (Melopsittacus undulatus) and other psittacine birds. Characteristic clinical signs include symmetric feather dystrophy and loss and deformity of the beak in some species (Ramis et al. 1998). Birds infected with PBFDV usually die as a result of sec- ondary infections, which support the view that they are immunosuppressed; bursal and thymic lesions are consistently detected (Todd 2000). Histopathology has been used for the routine diag- nosis of PBFD, but more recently, polymerase chain reaction (PCR) and haemagglutination (HA) tests have been described for the detection of PBFDV and haemagglutination inhibition (HI) for the detec- tion of antibodies to PBFDV (Riddoch, Raidal, & Cross 1996). Since all attempts to cultivate PBFDV 29 Onderstepoort Journal of Veterinary Research, 71:29–34 (2004) Psittacine beak and feather disease virus in budgerigars and ring-neck parakeets in South Africa J. ALBERTYN, K.M. TAJBHAI and R.R. BRAGG* Department of Microbial, Biochemical and Food Biotechnology, University of the Free State P.O. Box 339, Bloemfontein, 9300 South Africa ABSTRACT ALBERTYN, J., TAJBHAI, K.M. & BRAGG, R.R. 2003. Psittacine beak and feather disease virus in budgerigars and ring-neck parakeets in South Africa. Onderstepoort Journal of Veterinary Research, 71:29–34 Psittacine beak and feather disease (PBFD) is a common disease of the psittacine species and is caused by the psittacine beak and feather disease virus (PBFDV). In this study the occurrence of the disease in ring-neck parakeets and budgerigars in South Africa suffering from feathering problems, using polymerase chain reaction as a diagnostic test was investigated. The genetic variation between viral isolates was also studied. Results indicate that PBFDV can be attributed to being the cause of feathering problems in some of the ring-neck parakeets and budgerigars in South Africa. Genetic variation of isolates occurs between species and individuals. A cheap and easy to use method of blood sample collection on filter paper for diagnostic purposes was also evaluated. It proved to be less stressful to the birds and did not inhibit further processes. Keywords: Budgerigars, genetic variation, psittacine beak and feather disease virus, ring-neck para- keets, South Africa * Author to whom correspondence is to be directed. E-mail: BraggRR@sci.uovs.ac.za Accepted for publication 9 September 2003—Editor have so far been unsuccessful, the diagnosis of the disease must be confirmed by the demonstration of viral antigen or viral nucleic acid (Todd 2000). Bassami, Ypelaar, Berryman, Wilcox & Raidal (2001) studied the genetic diversity of PBFDV and concluded that there was no evidence that distinct- ly different genotypes occur although various differ- ences due to small deletions or insertions are pres- ent in the genomes of the isolates. They stated that the significance of these differences is unknown but that there is little evidence to support a relationship between genetic variation and regional distribution of isolates, or that there are differences in patho- genicity, antigenicity or any physiochemical charac- teristics of the virus. There is no known cure for PBFD but a vaccine consisting of inactivated PBFDV in a double-oil emulsion adjuvant system (DOE vaccine) has been investigated (Raidal et al. 1993; Raidal & Cross 1994), and its use can be a safe and effective aid for controlling the disease if it is combined with other management procedures, such as good biosecurity. In the disease, avian polyoma, caused by the avian polyomavirus (APV), clinical and pathological signs similar to those of PBFD are manifested. It has been known to occur in concurrent outbreaks with PBFD although documentation of these occurrences is limited (Latimer, Niagro, Campagnoli, Ritchie, Pesti & Steffens 1993; Ramis et al. 1998). Differentiation between the two diseases is difficult as the macro- and microscopical pathology of the two diseases is similar; diagnosis of avian polyoma disease is per- formed using techniques such as DNA in situ hybrid- ization or PCR (Ramis et al. 1998). Bird breeders in South Africa have been reporting feathering problems in budgerigars and have sus- pected that this may be due to APV infection. How- ever, Bragg (2002) reported that PBFDV is wide- spread in parrots of South Africa breeders and sug- gested that the feathering problems in the budgeri- gars could be due to PBFDV infection and not APV infection. This study was undertaken to determine if the occur- rence of PBFD in budgerigars and ring-neck para- keets in South Africa exists using PCR as an iden- tification tool. It was also undertaken to determine if there is any genetic variation between the isolates. A simple method for collecting blood samples on fil- ter paper was also evaluated for its use in down- stream processes for example the extraction of viral DNA followed by PCR. MATERIALS AND METHODS A total of seventeen birds were obtained from breeders in South Africa that showed feathering problems indicative of PBFDV infection. Blood sam- ples were collected in heparin lithium tubes from six ring-neck parakeets in Bloemfontein, five budgeri- gars in Pretoria and five in Cradock, South Africa (Table 1). In addition, the effectiveness of blood sample collection on filter paper was evaluated. A strip of Whatman No.1 filter paper of approximately 5 mm in width and 40 mm in length was sterilized by U.V. radiation. A wing vein of one ring-neck para- keet (P-BLM-4) was pricked with a sterile 0.33 mm diameter needle and four separate strips of filter paper was used to absorb blood resulting in the coverage of a blood-soaked area of approximately 5 x 5 mm in size at one end of the filter paper. The filter papers were air-dried vertically at room tem- perature followed by viral DNA extraction from the samples after 24, 48, 72 and 96 h. In addition, a blood spot sample from a ring-neck parakeet in George, South Africa (P-GEO) that was submitted on a commercially available specimen collection paper was similarly processed after one month of storage at –20 °C. Viral DNA was extracted from the blood samples using the QIAamp® DNA Blood Mini Kit and dried blood spot samples were processed using the QIAamp® DNA Mini Kit according to the manufac- turer’s instructions (Qiagen). The PCR amplifica- 30 Psittacine beak and feather disease virus in South Africa TABLE 1 Blood samples obtained from species and sources Sample Species Source P-BLM-1 Parakeet Bloemfontein P-BLM-2 Parakeet Bloemfontein P-BLM-3 Parakeet Bloemfontein P-BLM-4 Parakeet Bloemfontein P-BLM-5 Parakeet Bloemfontein P-BLM-6 Parakeet Bloemfontein P-GEO Parakeet George B-PTA-1 Budgerigar Pretoria B-PTA-2 Budgerigar Pretoria B-PTA-3 Budgerigar Pretoria B-PTA-4 Budgerigar Pretoria B-PTA-5 Budgerigar Pretoria B-CRA-1 Budgerigar Cradock B-CRA-2 Budgerigar Cradock B-CRA-3 Budgerigar Cradock B-CRA-4 Budgerigar Cradock B-CRA-5 Budgerigar Cradock tion was performed using a set of oligonucleotide primers [PB F1 (AACCCTACAGACGGCGAG-3’) and PB R1 (5’-GTCACAGTCCTCCTTGTACC-3’)] obtained from literature (Ypelaar et al. 1999; Bassami et al. 2001), amplifying a region of open reading frame 1 with a predicted size of 717 bp. Amplification was carried out according to Ypelaar et al. (1999) with a slight modification as follows; denaturation was carried out at 94 °C and anneal- ing was performed at 55 °C. A second set of prim- ers [PB F2 (5’-AACCATGCCGTCCAAGGA-3’) and PB R2 (5’-TATCAGTAATTGATGGGGTGGG-3’)] was designed to amplify the complete region of Open Reading Frame 1 of the PBFDV genome (GENBANK, Accession number AF080560) result- ing in an amplicon with a predicted size of 876 bp. Amplified products were identified by electrophore- sis on 0.8 % agarose gels and visualized under UV illumination. Both primer sets produced amplicons of the expected size and those obtained with the primer set PB F1 and PB R1 were used in subse- quent reactions. Restriction digests were performed to determine if there was any genetic difference between the isolates by means of digestion with the restriction endonuclease HaeIII. The digest prod- ucts were identified by electrophoresis on 2.5 % low melting point agarose gels and visualized under UV illumination together with a 50 bp step ladder (Pro- mega) as a molecular weight marker. Due to the possibility of different amplified products from sam- ples B-CRA-1, B-CRA-2, B-CRA-4 and B-CRA-5, these amplicons were cloned into pGEM™ TEasy Vector System (Promega), small scale plasmid iso- lation were performed, the inserts removed from two randomly selected clones and digested with HaeIII. The products were again visualized by elec- trophoresis. RESULTS All samples obtained from the ring-neck parakeets and budgerigars (except for sample B-CRA-3) pro- duced PCR amplicons of the expected size of approximately 700 bp, using primer set PB F1 and PB R1. Sample B-CRA-3, that consistently pro- duced a negative result, was further used as a neg- ative control The positive samples amplified with primer set PB F2 and PB R2 also produced frag- ments of the expected size of approximately 850 bp (data not shown). Amplicons obtained using primer set PB F1 and PB R1 were submitted to restriction digestion using HaeIII. The digest profile was similar for all the ring- neck parakeet samples obtained from Bloemfontein with approximate sizes of 450 bp, 250 bp and a third smaller fragment of approximately 50 bp (Fig. 1, lanes 2–7). The profile for sample P-GEO dif- fered in size from the other ring-neck parakeet samples yielding fragments of approximate sizes 460 bp, 250 bp and less than 50 bp (Fig. 1, lane 8). Samples obtained from the budgerigars in Pretoria (B-PTA-1, B-PTA-2, B-PTA-3, and B-PTA-5) had similar digest profiles with fragments of approxi- mately 410 bp, 225 bp and 50 bp in size (Fig. 2, lanes 2–4, 6), except for an additional fragment observed in sample B-PTA-4 with an approximate size of 370 bp (Fig. 2, lane 5). 31 J. ALBERTYN, K.M. TAJBHAI & R.R. BRAGG 450 bp 250 bp 150 bp 50 bp 1 2 3 4 5 6 7 8 9 FIG. 1 Restriction digest profile for the samples obtained from ring-neck parakeets using the restriction enzyme HaeIII. The digest products were loaded onto a 2.5 % agarose gel together with a 50 bp step ladder (Promega) [Lanes 1 and 9]. Samples P- BLM-1, P-BLM-2, P-BLM-3, P-BLM-4, P-BLM-5, P-BLM- 6 and P-GEO were loaded in lanes 2–8 respectively. Arrows on the right hand side indicate the discrepan- cy found of the ~50 bp band between the six samples obtained from Bloemfontein (upper arrow) in comparison to the sample obtained from George (lower arrow) 32 Psittacine beak and feather disease virus in South Africa 1 2 3 4 5 6 7 450 bp 250 bp 150 bp 50 bp FIG. 2 Restriction digest profile for the samples obtained from budgeri- gars (Pretoria) using the restric- tion enzyme HaeIII. The digest products were loaded onto a 2.5 % agarose gel together with a 50 bp step ladder (Promega) [Lanes 1 and 7]. The samples B- PTA-1, B-PTA-2, B-PTA-3, B- PTA-4 and B-PTA-5 (lanes 2–6 respectively) gave similar pro- files with sample B-PTA-4 con- taining an additional fragment of approximately 370 bp in size. The fragments below the 50 bp marker were light in intensity and therefore more difficult to observe 1 2 3 4 5 6 7 8 9 10 450 bp 250 bp 150 bp 50 bp 1 2 3 4 5 6 450 bp 250 bp 150 bp 50 bp FIG. 3 Restriction digest profile for the samples obtained from budgeri- gars (Cradock) using the restric- tion enzyme HaeIII showing mul- tiple fragments. The digest prod- ucts were loaded onto a 2.5 % agarose gel together with a 50 bp step ladder (Promega) [Lanes 1 and 6] as a molecular mass marker. The samples B- CRA-1, B-CRA-2, B-CRA-4 and B-CRA-5 were loaded in lanes 2–5 respectively FIG. 4 Restriction digest profile for two randomly picked clones from each sample obtained from budgerigars (Cradock) using the restriction enzyme HaeIII. The digest products were loaded onto a 2.5 % agarose gel togeth- er with a 50 bp step ladder (Pro- mega) [Lanes 1 and 10] as a molecular mass marker. Load- ing was performed as follows, B- CRA-1 [lanes 2 and 3], B-CRA-2 [lanes 4 and 5] B-CRA-4 [lanes 6 and 7] and B-CRA-5 [lanes 8 and 9] The digest profile obtained for the samples obtained from budgerigars in Cradock continuously resulted in an unusual profile (Fig. 3) that led to the possi- bility that more than one PCR amplicon (i.e. ampli- cons from genetically different viral isolates) of approximately 700 bp was present. These amplicons were cloned into the vector pGEM™ TEasy, inserts were purified from two ran- domly selected clones for each sample, and digest- ed with HaeIII (Fig. 4). Clones from B-CRA-1 (Fig. 4, lanes 2 and 3) were similar in profile to each other but different from B-CRA-2 and B-CRA-4 [Fig. 4, lanes 4–7 respectively], which in turn shared a similar profile. Sample B-CRA-5 [Fig. 4, lanes 8 and 9] had a different profile for each clone; with one clone (Fig. 4, lane 8) sharing a similar profile with samples B-CRA-2 and B-CRA 4 and the second clone (Fig. 4, lane 9) sharing a profile with that of B- CRA-1 This profile indicates that at least two genet- ically different PBFDV isolates were present in these birds and, more importantly, that at least one bird contained both of these genetically different isolates. The samples collected on filter paper all produced amplicons of the expected size (approximately 700 bp) and all amplicons, including the blood sample that was processed after one month of storage at –20 °C, showed the same intensity (Fig. 5). None of the products showed any non-specific amplification indicating that the filter paper collection methods is an easy and cost effective way to collect and pro- cess blood samples. DISCUSSION The PCR primer set PB F1 and PB R1 and primer set PB F2 and PB R2 amplified the expected prod- ucts in all the samples tested except for one budgerigar that tested negative. The PCR ampli- cons obtained by amplification using primer set PB F2 and PB R2 were not used in further processes although this primer set also resulted in reproduc- ible results. The reason for designing these primers and performing amplifications using them was to attempt to amplify the entire ORF1, which will be important for future studies and the potential devel- opment of a subunit vaccine. The variation in the digest profiles between species and individuals suggests genetic variation in the sequences of the amplified region but it is still un- known if these variances indicate any differences in antigenicity or pathogenicity. Profiles for all the samples obtained from the different regions in the country were not similar to each other even though they were from the same species (budgerigars from Pretoria and Cradock). There may be a possibility that there is a relationship between regional distri- bution and genetic variation but this study does not give enough evidence to confirm or refute this. Bassami et al. (2001) reported that there is a pos- sibility that adaptation of particular genotypes to specific species may occur and that regional differ- ences in strains may develop; they suggested that these differences would have a significant implica- tion of the development of a universal PCR assay that would detect all strains of PBFDV. The genet- ic variation between these birds lends an important view in the production of a vaccine. If there are sig- nificant differences in the antigenicity or patho- genicity and other physiochemical characteristics of these isolates then it is important to develop a vaccine that can effectively protect a bird against any isolate or isolates that may infect it at any par- 33 J. ALBERTYN, K.M. TAJBHAI & R.R. BRAGG 1 2 3 4 5 6 7 8 450 bp 250 bp 150 bp 50 bp FIG. 5 PCR amplified products of PBFDV (P- BLM-4) from samples collected on fil- ter paper, followed by DNA extraction after 24 [lane 2], 48 [lane 3], 72 [lane 4] and 96 h [lane 5]. Lane 6 contains the PCR product of a sample (P- GEO) that was received on commer- cially available specimen collection paper and processed after 1 month. Lanes 1 and 8, contains a molecular mass marker (λ EcoR1/HindIII), and lane 7 a negative control from a sam- ple that was shown to be negative (B- CRA-3) ticular time. In the related porcine circovirus (PCV) two distinct genotypes have been identified (Todd 2000) and it is possible that similar genotypic dif- ferences have occurred in PBFDV. The PCR tests performed were reproducible and confirm the occurrence of PBFDV infection in the ring-neck parakeets and budgerigars in the regions from where the samples were obtained. However, it was difficult to rule out the possibility of APV infec- tion in the birds tested. Although a PCR test using primers to amplify the region coding for protein VPI of APV was performed and negative results were obtained, these results are not conclusive due to the lack of a positive control. A positive control was difficult to obtain as there were no reported cases of APV infection in South Africa at the time of study. This study then does not exclude the possibility of a concurrent infection of PBFDV and APV in the birds tested. The method evaluated in this study for the collec- tion of blood using filter paper was successful and did not have any negative effects on downstream processes. The results were reproducible and the PCR amplification was comparable to that per- formed using frozen blood samples. This method is simple, easy, does not require large amounts of blood (thus less stressful to the birds) and the sam- ples do not require the use of anticoagulants, do not have to be frozen and are easy to transport. Most importantly however, it does not affect down- stream processes. With all these advantages it is evident that the implementation of this method of sample collection is a suitable replacement to the more traditional methods of blood collection. In conclusion, this study has shown the occurrence of PBFDV infection in ring-neck parakeets and budgerigars in South Africa by establishing and optimising a reliable PCR diagnostic test with reproducible results. The primers that were designed during this project were successful in producing the amplicon of expected size when used in the PCR test. Genetic variation between isolates from ring-neck parakeets and budgerigars is evident as well as genetic differences between isolates within individuals of the same species. However, the significance of these differences to PBFDV antigenicity, pathogenicity and other phys- iochemical characteristics must be investigated if an effective vaccine is to be produced. Blood sam- ple collection on filter paper is easy to use, is less stressful to the birds and does not impact negatively on further processes. ACKNOWLEDGEMENTS We appreciate the help of Michel Labuschagne of the University of the Free State in the practical work. We are grateful to Sam Rosslee, Pierre Swart and G. B. Swart for supplying blood samples and to Dr F. Potgieter of the University of the Free State for his assistance with the care of the birds. REFERENCES BASSAMI, M.R., YPELAAR, L., BERRYMAN, D., WILCOX, G.E. & RAIDAL, S.R. 2001. Genetic diversity of beak and feather disease virus detected in psittacine species in Australia. Virology, 279:392–400. BRAGG, R. 2002. Feather disorders in budgerigars in South Africa. The Budgerigar Society of South Africa, Quarterly Bulletin, 6–7. LATIMER, K.S., NIAGRO, F.D., CAMPAGNOLI, R.P., RITCHIE, B.W., PESTI, D.A. & STEFFENS, W.L. 1993. Diagnosis of concurrent avian polyomavirus and psittacine beak and feather disease virus infections using DNA probes. Journal of the Association of Avian Veterinarians, 7:141–146. PASS, D.A., PLANT, S.L. & SEXTON, N. 1994. Natural infection of wild doves (Streptopelia senegalensis) with the virus of psittacine beak and feather disease. Australian Veterinary Journal, 71:307–308. RAIDAL, S.R., FIRTH, G.A. & CROSS, G.M. 1993. Vaccination and challenge studies with psittacine beak and feather dis- ease virus. Australian Veterinary Journal, 70:437–441. RAIDAL, S.R. & CROSS, G.M. 1994. Control by vaccination of psittacine beak and feather disease in a mixed flock of Agapornis spp. Australian Veterinary Practice, 24:178–180. RAMIS, A., LATIMER, K.S., GIBERT, X. & CAMPAGNOLI, R. 1998. A concurrent outbreak of psittacine beak and feather disease virus, and avian polyomavirus infection in budgeri- gars (Melopsittacus undulatus). Avian Pathology, 27:43–50. RIDDOCH, P.A., RAIDAL, S.R. & CROSS, G.M. 1996. Psittacine circovirus antibody detection and an update on the methods for diagnosis of psittacine beak and feather dis- ease. Australian Veterinary Practice, 26:134–139. SEXTON, N., PENHALE, W.J., PLANT, S.L. & PASS, D.A. 1994. Use of goose red blood cells for detection of infection with psittacine beak and feather disease virus by haemag- glutination and haemagglutination inhibition. Australian Veterinary Journal, 71:345–347. STUDDERT, M.J. 1993. Circoviridae: new viruses of pigs, par- rots and chickens. Australian Veterinary Journal, 70:1221– 122. TODD, D. 2000. Circoviruses: immunosuppressive threats to avian species: a review. Avian Pathology, 29:373–394. YPELAAR, I., BASSAMI, M.R., WILCOX, G.E. & RAIDAL, S.R. 1999. A universal polymerase chain reaction for the detec- tion of psittacine beak and feather disease virus. Veterinary Microbiology, 68:141–148. 34 Psittacine beak and feather disease virus in South Africa