DETECTION OF BEAK AND FEATHER DISEASE VIRUS AND AVIAN POLYOMAVIRUS DNA IN PSITTACINE BIRDS IN POLAND

141 Bull Vet Inst Pulawy 54, 141-146, 2010 DETECTION OF BEAK AND FEATHER DISEASE VIRUS AND AVIAN POLYOMAVIRUS DNA IN PSITTACINE BIRDS IN POLAND TOMASZ PIASECKI AND ALINA WIELICZKO Department of Epizootiology with Clinic of Birds and Exotic Animals, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, 50-360 Wroclaw, Poland tomasz.piasecki@up.wroc.pl Received for publication November 10, 2009 Abstract The first survey on the incidence of beak and feather disease virus (BFDV) and avian polyomavirus (APV) among the captive psittacine birds in Poland was presented in this study. Samples were collected between 2006 and 2009. A total number of 751 symptom free birds from 31 genera and 59 species were tested for BFDV and APV by means of a PCR assay. BFDV was found in 25.3% and APV in 22.24% of the analysed population. The co-existence of BFDV and APV was noted in 5.99% of the birds. Key words: psittacine birds, avian polyomavirus, psittacine beak and feather virus, PCR. Beak and feather disease virus (BFDV) was first identified in Australia in the early 1980s in wild populations of cockatoos (14). It is classified together with the chicken anaemia virus and other avian circoviruses (pigeon circovius - PiCV, goose circovirus - GCV, canary circovirus - CaCV) in the genus Circovirus of the Circoviridae family. BFDV is a non-enveloped virus of 14 17 nm diameter with isometric or spherical symmetry. Its genome contains a circular singlestranded DNA (ssdna), approximately 2,000 bp (1, 11). An analysis of the replicative form of the virus demonstrated seven open reading frames (ORFs). Three of them were identified in the virion strand and four ORFs were found in the replicative strand. These ORFs potentially encode viral proteins (1). BFDV is the most common viral pathogen of parrots and its incidences have been confirmed in more than 60 psittacine species (19). Nowadays, this infection is widespread in wild populations of cockatoos and rosellas in Australia and Indonesia (17, 18). The reason for current presence of BFDV in all continents is the international trade of parrots. Psittacine beak and feather disease (PBFD) is a chronic, progressive and irreversible viral disease, characterised by a progressive symmetrical feather dystrophy and loss, occasionally associated with beak deformities (17, 18). Avian polyomavirus (APV) was first characterised as a pathogen in young budgerigars (Melopsittacus undulatus) in 1981 (4). It was designated budgerigar fledgling disease virus but now it is called APV because of its broad host range. APV is included in the genus Polyomavirus family Papovaviridae. It has an icosahedral, non-enveloped capsid of about 40-50 nm diameter. The APV genome is a circular doublestranded DNA, 4,981 bp in size. The main clinical symptoms of APV infection include hepatitis, ascites, and hydropericardium. Its mortality rate reaches up to 100% in fledglings (8). Sometimes APV infection may result in peracute death with subclinical signs only. In budgerigars APV is a causative agent of budgerigar fledgling disease (BFD), which is characterised by feather abnormalities and loss of fledglings and young budgerigars. At a cellular level, APV induces enlarged clear basophilic nuclear inclusions. They are found mostly in the liver and spleen, although may also occur in other organs and tissues (2). Material and Methods Seven hundred and fifty-one parrots of 59 species obtained from private Polish breeders were subjected to this study. All the birds were healthy on clinical inspection. The material was divided into three groups depending on the size of aviary: group 1 included subjects from amateur aviaries of 1 to 3 birds, group 2 from 4- to 10-bird aviaries, and group 3 from professional aviaries of more than 10 birds. DNA was isolated from feathers (6 8 pcs) obtained from sternal and hypogastrial region using Genomic DNA Prep Plus columns (A&A Biotechnology, Poland) in accordance with

142 manufacturer s instructions. Next, the obtained DNA was analysed using PCR technique. PCR reaction for psittacine beak and feather disease virus (PBFDV). In order to detect BFDV, PCR was performed to amplify ORF1 fragment of 717 bp in length, as described by Ypelaar et al. (20). The sequence of primers was as follows: P 2-5 -AAC CCT ACA GAC GGC GAG-3 (182-199) and P 4-5 -GTC ACA GTC CTC CTT GTA CC-3 (879-898). PCR amplifications were carried out in MJ Mini Personal Thermal Cycler BioRad in a total volume of 25 µl containing: 100 ng of DNA template, 1 U of REDTaq DNA Polymerase (Sigma), 1 x PCR buffer (10x REDTaq PCR Reaction Buffer, Sigma), 200 µm dntp mixture (Sigma), and 25 pmol of each primer. Amplification conditions were: 96 C for 5 min, 32 cycles of 96 C for 30 s, 60 C for 30 s, 72 C for 90 s, and a final extension at 72 C for 5 min. The resulting products of 717 bp were separated on 2% agarose gels with ethidium bromide. PCR reaction for APV. PCR amplification of a 310 bp fragment of the APV genome was performed using primers described by Johne and Müller (7). The sequence of primers was as follows: 5 -CAA GCA TAT GTC CCT TTA TCC C-3 (4303-4324) and 5 -CTG TTT AAG GCC TTC CAA GAT G -3 (4,612-4,591). PCR amplifications were carried out in a total volume of 25 µl containing: 100 ng of DNA template, 1 U of REDTaq DNA Polymerase (Sigma), 1 x PCR buffer (10x REDTaq PCR Reaction Buffer, Sigma), 200 µm of 10 mm dntp mixture (Sigma), and 25 pmol of each primer. Amplification conditions were 95 C for 5 min, 35 cycles of 94 C for 30 s, 60 C for 30 s, 72 C for 30 s, and a final extension at 72 C for 10 min. The resulting products of 310 bp were separated on 2% agarose gels with ethidium bromide. Results The studies were carried out between 2006 and 2009 and included a total number of 751 parrots belonging to 31 genera and 59 species. The analysed birds originated from 113 small amateur aviaries (n=113), 47 medium ones (n=107), and 29 large professional ones (n=531). Four hundred and fifty-seven subjects belonged to the Old World genera (Europe, Africa, Asia, Australia, and Oceania), whereas the remaining 294 parrots to the New World ones (North, Central and South America) (Tables 1, 2, 3). The PBFD DNA was found in 190 examined birds, which corresponds to 25.3%, whereas APV DNA was detected in 167 subjects, which comprises to 22.24%. The co-existence of genetic material of both tested pathogens occurred in 45 birds, i.e. in 5.99% (Table 1). Psittacine beak and feather disease virus. The incidence of PBFDV infections in parrots was analysed in relation to the size of aviary. The fraction of PBFDV-infected parrots from amateur breedings amounted to 22.12%, whereas it comprised 25.23% and 25.99% for medium and large aviaries, respectively. If analysed per breeding, the incidence of PBFDV was noted in 22.12%, 40.43% and 75.86% of small, medium and large aviaries, respectively (Table 2). Finally, the incidence of PBFDV infections was related to geographic origin of genera tested. It was noted that the fractions of positive birds from the New and Old World amounted to 17.35% and 30.42%, respectively (Table 3). Avian polyomavirus. APV DNA was found in 23.01% of parrots from amateur breedings and in 23.36% and 21.85% ones from medium and large aviaries, respectively. These figures corresponded to the infection of 23.01%, 34.04%, and 58.62% of small, medium, and large aviaries, respectively (Table 2). The incidence of APV was similar in New and Old World genera and amounted to 20.07% and 23.63%, respectively (Table 3). Discussion Beak and feather disease is known worldwide for nearly 40 years. Nonetheless, it still results in losses of either the captive parrots or naturally living ones (5, 10). Prophylactic screening and elimination of positive birds from the stock seem to be the most important steps towards the limitation of the infection spread. The first screenings in Europe were performed in Italy and Germany between 2000 and 2004 (2, 15). The German study performed by Rahaus and Wolff (15) on 146 clinically healthy parrots revealed that the circovirus was present in 39.2% of the tested population. Hence, that fraction was markedly higher than the one from our study. The fraction of positive birds in Italy, in turn, was markedly lower and amounted to 8.05% of 1,516 parrots tested (2). We think, that the mentioned differences are probably related to various types of material used for testing. We and the German authors utilised parrots feathers as a source of DNA for further analyses. In the Italian study, however, DNA was isolated from psittacine blood (2). Consequently, any comparisons among the aforementioned results are irrelevant due to methodological differences. Our data regarding the frequency of positive birds were similar to the results presented by Hsu et al. from Taiwan (6), who found PBFDV in 41.2% of 165 parrots tested. Likewise in our study, the analyses were performed on psittacine feathers. In American study, in turn, performed in the 1990s on the DNA isolated from parrots blood, the frequency of PBFD infections amounted to 5%. Since the results were considered very high, further monitoring of PBFDV infections was performed, which in turn, showed a decrease in the number of infected parrots the fraction of circovirus-positive birds dropped to 3.5% after 5 years from the initial study (3).

143 Genus Table 1 Incidence of PBFDV and APV infections in various psittacine species from Poland Species birds tested PBFDV positive birds APV positive birds PBFDV and APV positive birds Agapornis Agapornis sp. 10 6 6 5 Alisterus Alisterus scapularis 7 4 1 0 Amazona aestiva 36 6 9 0 Amazona amazonica 25 2 4 0 Amazona autumnalis 5 0 0 0 Amazona barbadensis 8 3 6 2 Amazona brasiliensis 1 0 0 0 Amazona Amazona farinosa 5 0 1 0 Amazona festiva 7 0 0 0 Amazona finschi 4 0 0 0 Amazona leucocephala 4 0 1 0 Amazona ochrocephala 14 2 2 0 Amazona oratrix 4 0 0 0 Amazona sp. 23 2 2 0 Anodorhynchus Anodorhynchus hyacinthinus 1 0 0 0 Aprosmictus Aprosmictus erythropterus 4 4 0 0 Ara ararauna 72 17 9 2 Ara chloroptera 26 8 3 0 Ara Ara hybryda 3 0 2 0 Ara macao 6 3 3 1 Ara militaris 2 0 1 0 Aratinga acuticaudata 1 1 0 0 Aratinga Aratinga jandaya 4 0 4 0 Aratinga solstitialis 5 0 3 0 Barnardius Barnardius barnardi 9 8 0 0 Barnardius zonarius 2 2 0 0 Cacatua alba 2 0 1 0 Cacatua ducorpsii 2 0 0 0 Cacatua Cacatua goffini 4 0 0 0 Cacatua moluccensis 6 0 1 0 Cacatua sp. 1 1 0 0 Cacatua sulphurea 1 0 0 0 Chalcopsitta Chalcopsitta duivenbodei 2 0 0 0 Cyanoliseus Cyanoliseus patagonus 4 1 2 1 Cyanoramaphus Cyanoramaphus auricepis 4 0 0 0 Diopsittaca Diopsittaca nobilis 5 2 3 0 Eclectus Eclectus roratus 13 1 4 2 Eolophus Eolophus roseicapillus 28 8 5 3 Forpus Forpus coelestis 2 1 0 0 Melopsittacus Melopsittacus undulatus 11 7 2 2 Myiopsitta Myiopsitta luchsi 1 0 0 0 Nymphicus Nymphicus hollandicus 2 0 0 0 Orthopsittaca Orthopsittaca manilata 7 0 0 0 Pionites Pionites melanocephala 15 1 3 0 Platycercus Platycercus elegans 2 2 0 0 Platycercus eximius 4 3 0 0 Poicephalus Poicephalus robustus 2 1 1 0 Poicephalus senegalus 25 6 3 0 Polytelis Polytelis alexandre 1 0 0 0 Polytelis swainsonii 2 0 1 0 Probosciger Probosciger aterrimus 2 1 0 0 Propyrrhura Propyrrhura maracana 3 2 1 1 Psephotus Psephotus haematonotus 3 2 2 1 Psittacula alexandri 3 3 0 0 Psittacula cyanocephala 2 2 0 0 Psittacula Psittacula debrina 6 1 0 0 Psittacula eupatria 10 1 5 0 Psittacula krameri 48 20 17 5 Psittacus Psittacus erithacus 219 53 49 18 Pyrrhura Pyrrhura sp. 1 0 0 0 Trichoglossus Trichoglossus haematodus 20 3 10 2 Total number (%) 751 190 (25.30) 167 (22.24) 45 (5.99)

144 Table 2 Incidence of PBFDV and APV infections in parrots depending on the size of aviary of their origin Size of aviary Small (amateur) (1 3 birds) Medium (4 10 birds) Large (professional) (more than 10 birds) birds tested 113 107 531 Total 751 Number (%) of birds infected aviaries Number (%) of aviaries infected PBFDV APV tested PBFDV APV 25 (22.12) 27 (25.23) 138 (25.99) 190 (25.30) 26 (23.01) 25 (23.36) 116 (21.85) 167 (22.24) 113 47 29 188 25 (22.12) 19 (40.43) 22 (75.86) 66 (35.11) Table 3 Incidence of PBFDV and APV infections in parrots depending on the geographical origin of their genera Genus birds tested Old World genera Number (%) PBFDV APV PBFDV/APV Agapornis 10 6 (60.0) 6 (60.0) 5 (50.00) Poicephalus 27 7 (25.93) 4 (14.81) 0 (0.0) Psittacula 69 27 (39.13) 22 (31.88) 5 (7.25) Psittacus 219 53 (24.2) 49 (22.37) 18 (8.22) Alisterus 7 4 (57.14) 1 (14.29) 0 (0.0) Aprosmictus 4 4 (100) 0 (0.0) 0 (0.0) Barnardius 11 10 (90.91) 0 (0.0) 0 (0.0) Cacatua 16 1 (6.25) 2 (12.50) 0 (0.0) Chalcopsitta 2 0 (0.0) 0 (0.0) 0 (0.0) Cyanoramaphus 4 0 (0.0) 0 (0.0) 0 (0.0) Eclectus 13 1 (7.69) 4 (30.77) 2 (15.38) Eolophus 28 8 (28.57) 5 (17.86) 3 (10.71) Melopsittacus 11 7 (63.64) 2 (18.18) 2 (18.18) Nymphicus 2 0 (0.0) 0 (0.0) 0 (0.0) Platycercus 6 5 (83.33) 0 (0.0) 0 (0.0) Polytelis alexandre 3 0 (0.0) 1 (33.33) 0 (0.0) Probosciger 2 1 (50.0) 0 (0.0) 0 (0.0) Psephotus 3 2 (66.67) 2 (66.67) 1 (33.33) Trichoglossus 20 3 (15.0) 10 (50.00) 2 (10.00) 139 TOTAL 457 (30.42) New Word genera 108 (23.63) 38 (8.32) Amazona 136 15 (11.03) 25 (18.38) 2 (1.47) Anodorhynchus 1 0 (0.0%) 0 (0.0) 0 (0.0) Ara 109 28 (25.69) 18 (16.51) 3 (2.75) Aratinga 10 1 (10.00%) 7 (70.0) 0 (0.0) Cyanoliseus 4 1 (25.00%) 2 (50.0) 1 (25.0) Diopsittaca 5 2 (40.00%) 3 (60.0) 0 (0.0) Forpus 2 1 (50.00%) 0 (0.0) 0 (0.0) Myiopsitta 1 0 (0.0%) 0 (0.0) 0 (0.0) Orthopsittaca 7 0 (0.0%) 0 (0.0) 0 (0.0) Pionites 15 1 (6.67%) 3 (20.0) 0 (0.0) Propyrrhura 3 2 (66.67%) 1 (33.33) 1 (33.33) Pyrrhura 1 0 (0.0%) 0 (0.0) 0 (0.0) TOTAL 294 51 (17.35) 59 (20.07) 7 (2.38) 26 (23.01) 16 (34.04) 17 (58.62) 59 (31.38)

145 Several studies showed that the New World genera are significantly less susceptible to PBFD infections (2, 17). That finding was generally confirmed by the results of presented study. Our conclusion was different; however, when the incidence of PBFDV was compared among three of the most frequently infected species. PBFDV was revealed in 24.2% of the African Grey parrots (Psittacus erithacus) as the representatives of the Old World, but only in 11.03% of the Amazon parrots (Amazona sp.) and in 25.69% of the Aras (Ara sp.) representing New World. Consequently, the Amazon parrots seem to be less susceptible to PBFDV infection in comparison to other New World parrots. Recently, the problem of psittacine beak and feather disease is increasing in Africa. According to Heath et al. (5) 10% to 20% of fledglings from South African aviaries dies every year due to PBFD infections. The disease occurs endemically in that region of Africa, and its frequency increases among naturally living species of parrots, and particularly in Poicephalus robustus and Agapornis nigrigenis. Either the parrots breed in the South African aviaries, or those obtained from their natural environment, are imported mainly to the European markets and consequently may constitute an important component of epizootic chain of disease discussed. Infections with APV were the most frequently described in budgerigars, where they resulted in budgerigar fledgling disease (9, 10). The remaining psittacine species exhibit variable susceptibility to that infection (17). A fraction of positive results found in present experiment (22.24%) is surprisingly high compared to the ones reported by previous studies performed in Europe. Bert et al. (2) revealed APV infection in only 0.79% birds from Italy, whereas Rahaus and Wolff (16) did not found APV in any of 85 parrots tested in Germany. Those differences may be related to the regions of APV genome used for diagnostics. In the presented study, we have amplified 310 bp fragment of the APV genome using primers located in the t/t antigen region. In German and Italian studies, in turn, the 550 bp VP1 fragments of the APV genome were amplified (2, 16). The proportional distribution of the results, however, was totally different in studies performed in Asia. Ogawa et al. (12, 13), whose diagnostics were based on the t/t antigen region revealed, that APV infection occurred in 2.7% of parrots from Japan. Hsu et al. (6), in turn, who used the same protocols as Bert et al. (2) and Rahaus and Wolff (16) obtained 15.2% of the positive results. Most of the parrots tested in the latter study, however, exhibited symptoms characteristic for APV and/or PBFD infections (6). In conclusion, high fraction of parrots from Poland, infected with PBFD and/or APV, seems to be related to numerous factors. 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