Advance Publication
The Journal of Veterinary Medical Science
Accepted Date: 29 Oct 2012 J-STAGE Advance Published Date: 12 Nov 2012 1 Relatedness between host species and genotype of beak and feather disease virus
2 suggesting possible interspecies cross infection during bird trade
3
4 Hirohito Ogawa1†, Rajesh Chahota1#, Kenji Ohya2, Tsuyoshi Yamaguchi3, and Hideto
5 Fukushi1,2*
6
7 1 Department of Applied Veterinary Sciences, United Graduate School of Veterinary
8 Sciences, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan
9 2 Laboratory of Veterinary Microbiology and Infectious Diseases, Faculty of Applied
10 Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan
11 3 The Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University,
12 4-101 Yanagido, Koyama Minami, Tottori 680-8550, Japan
13
14 † Present address: Hokkaido University Research Center for Zoonosis Control, North
15 20, West 10 Kita-ku, Sapporo 001-0020, Japan
16 # Present address: Department of Veterinary Microbiology
17 College of Veterinary and Animal Sciences
18 CSK HP Agricultural University, Palampur, H.P. 176 062 INDIA
1 19 * Corresponding author: Hideto Fukushi
20 Laboratory of Veterinary Microbiology and Infectious Diseases, Faculty of Applied
21 Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan
22 Tel and Fax: +81-58-293-2946
23 E-mail: [email protected]
24
25 Running head: RELATEDNESS BETWEEN HOST AND GENOTYPE OF BFDV
26
27
2 28 ABSTRACT
29 Beak and feather disease virus (BFDV) is a causative agent of psittacine beak and
30 feather disease (PBFD), which shows a characteristic feather disorder in psittacine birds.
31 In the present study, the subclinical infection rate of PBFD in imported and domestically
32 bred psittacine birds was investigated by polymerase chain reaction. As a result, 126 of
33 402 birds (31.3 %) were found to be BFDV positive. The DNA sequences of the part of
34 open reading frame (ORF) C1 were determined for 16 BFDV-positive randomly selected
35 samples. One of 16 samples was found to have a mixed infection, and 5 different BFDV
36 sequences were obtained from a single African grey parrot. In phylogenic analysis, almost
37 BFDV sequences included in each genetic cluster of phylogenic tree belonged to the same
38 psittacine subfamily. BFDV derived from African grey parrot was closely related to the
39 BFDV derived from cockatoos by way of exception. The natural habitat of the African
40 grey parrot and cockatoos is different, therefore the possibility of interspecies cross
41 infection through the bird trade is suggested from the exceptional BFDV sequences.
42 KEY WORDS
43 Circovirus; epidemiology; phylogenic analysis; Psittacine beak and feather disease
44
3 45 Psittacine beak and feather disease (PBFD) is a specific disease in psittacine birds
46 [14]. PBFD shows a sudden death in peracute or acute forms and characteristic feather
47 disorder in chronic form. Disease control is the most important because of no etiological
48 treatment, therefore early detection is required. PBFD is caused by a beak and feather
49 disease virus (BFDV) in which the virion has a diameter of 14 - 17 nm and a
50 nonenveloped icosahedral with a complete genome size of approximately 2 kb
51 single-stranded circular DNA [2]. BFDV is a member of the genus Circovirus in the
52 family Circoviridae [1, 9]. The genome contains two major open reading frames (ORFs),
53 encoding the replication-associated protein (V1) and the capsid protein (C1).
54 PBFD has been confirmed worldwide [3, 7, 12]. Genomic analyses of BFDV have
55 been advanced in recent years, and genetic diversity in ORF V1 and C1 has been also
56 reported [2, 5, 6, 8]. Epidemiological studies are still less than other studies, i.e.
57 pathological or etiological studies, although some studies were recently reported in some
58 countries [3, 7, 12]. Furthermore, there are also no reports for other countries such as
59 Singapore and Indonesia, which act as the main bases of the bird trade and have many
60 psittacine breeding farms. Therefore, it is necessary epidemiological studies including
61 these countries, and molecular analyses to trace the roots of BFDV genotypes among
62 countries and the relatedness between BFDV sequences and host species as in previous
4 63 reports [5, 6, 8, 15].
64 In this study, we investigated the subclinical infection rate of PBFD in 402 live
65 psittacine birds imported from some countries and bred in Japan from April, 2003 to
66 March, 2004. The details of these birds have been described in our previous study [10].
67 DNA was extracted from the blood or feathers with a SepaGene nucleic acid extraction kit
68 (Sanko Junyaku Co., Tokyo, Japan) according to the manufacturer’s instructions [10].
69 PCR for BFDV examination was performed using Primer2 (5'-AAC CCT ACA GAC GGC
70 GAG-3') and Primer4 (5'-GTC ACA GTC CTC CTT GTA CC-3') [13], which targets a
71 part of ORF V1, to compare with other epidemiological reports [3, 7, 12]. PCR was
72 carried out by using TaKaRa Ex Taq (TaKaRa Bio., Shiga, Japan) as described previously
73 [11, 18]. In our laboratory, the detection limit of this PCR was 2.44x104 copies of viral
74 DNA per 50 l reactions.
75 A total of 126 BFDV-positive birds (31.3% of the birds) were found in eleven genera
76 (Table 1). Exporting countries of BFDV-positive birds were widely distributed, although
77 no BFDV-positive birds were detected from European countries and Guyana (Tables 1 and
78 2).
79 Sixteen BFDV samples were randomly chosen for sequencing to investigate the
80 relationship among the BFDV. DNA samples were amplified with PBFDdupF (5'-TTG
5 81 GGT CCT CCT TGT AGT GGG ATC-3') and PBFDdupR (5'-CAG ACG CCG TTT CTC
82 AAC CAA TAG-3'), which were used to amplify a 495-bp fragment corresponding to the
83 part of ORF C1 [14], because genetic clusters were accorded to some psittaicne species or
84 subfamily in the phylogenic analysis using BFDV ORF C1 [5, 6, 11, 13, 18]. Molecular
85 cloning and sequencing were examined as described previously [11], and at least three
86 clones were sequenced. Sequences were edited using Genetyx-Mac version 13 computer
87 software (Genetyx Co., Tokyo, Japan) and assembled using Genetyx-Mac/ATSQ version
88 4.2.4 computer software (Genetyx Co.). ClustalX 1.82 [17] and PHYLIP version 3.6 were
89 used for multiple sequence alignment and phylogenic analysis of sequences. A phylogenic
90 analysis of nucleotide sequences using the maximum-likelihood was conducted using a
91 DNAML. Fifteen sequences of 16 samples were determined without difficulty, however,
92 the other 1 sample derived from an African grey parrot (Psittacus erithacus erithacus)
93 produced a mixture of several sequences. The PCR product derived from the above
94 African grey parrot was cloned again, and 5 clones, which were randomly chosen, were
95 independently sequenced. Five sequences determined (AGP-SA1P-1 to AGP-SA1P-5) had
96 several nucleotide diversities. Thus, we found at least 5 BFDV sequences in a single
97 African grey parrot, and 15 sequences (AGP-SA2P, TGP-SA1P, EC-SG1P, WC-SG1P,
98 WC-IN1P, YCC-IN3P, YCC-IN4P, BWP-SA1P, BP-SA1P, BHP-SA1P, RBP-SA1P,
6 99 SP-SA1P, AP-UN1P, EP-JP1P, and DP-SA1P) were determined from 16 BFDV samples
100 (Table 3). Accession numbers of sequences presented in this study are AB277726 to
101 AB277745. A total of 20 sequences read in the present study were compared with 29
102 sequences published in DNA databases (Table 3).
103 The length of each nucleotide sequence was 447-bp long except for 7 sequences that
104 were 444 bp including TGP-SA1P derived from a timneh grey parrot (Psittacus erithacus
105 timneh), MMC-WA derived from a major mithell’s cockatoo (Cacatua leadbeateri),
106 SCC-NT derived from a sulphur-crested cockatoo (Cacatua galerita), and PEP01-POR b,
107 AFG3-ZA, AGP-SA1P-2 and AGP-SA1P-4 derived from African grey parrots. The
108 homology of nucleotide and deduced amino acid sequences were from 80.8% to 100% and
109 from 73.6 % to 100%, respectively.
110 The phylogenic clusters were defined as any groups of sequences at least 5%
111 divergent from the next closest cluster according to Heath et al. [6], and 17 clusters were
112 identified in the phylogenic tree (Fig. 1). Host birds of clusters I to V were mainly
113 Cacatuinae. Host birds of clusters VI and VII were Lorinae. All host birds of cluster VIII
114 to XVII were Psittacinae. Although most sequences were specific for host species, the
115 sequences of BFDV derived from the African grey parrot were found in many clusters.
116 There is no regularity between genetic clusters and geographic origin of host birds. It has
7 117 been reported that genetic clusters were accorded to some psittaicne species or subfamily
118 in the phylogenic analysis using BFDV ORF C1 [5, 6, 11, 13, 18]. Contrary to previous
119 reports, 3 sequences (AFG4-ZA, AGP-SA2P and AGP-SA1P-3) derived from the African
120 grey parrot were found in the genetic clusters I, II and V, clusters mainly comprised of
121 Cacatuinae (Fig. 1).
122 PBFD was first described in various Australian cockatoos [14]. The natural habitat of
123 the African grey parrot and birds in Cacatuinae are Africa and Australasia, respectively [4,
124 16]. Fundamentally, birds in Cacatuinae are not able to make contact with the African
125 grey parrot except through the bird trade. Heath et al. [6] reported that BFDV in Africa
126 represented distinctly unique genotypes, and they suggested that Australian and African
127 BFDV populations have possibly diverged sufficiently to produce regionally distinct
128 lineage. It is conceivable that BFDV derived from the African grey parrot and birds in
129 Cacatuinae has usually evolved into unique genotypes, respectively. However, in the
130 present study, not only the sequences of AGP-SA2P and AGP-SA1P-3, but also the other
131 BFDV sequences derived from South Africa have no distinctly unique genotype lineage.
132 Therefore, the bird trade was likely to cause contact between the African grey parrot and
133 birds in Cacatuinae, and subsequently, the African grey parrot was infected with BFDV
134 derived from the birds in Cacatuinae.
8 135 In conclusion, we represented a wide-area molecular epidemiology of PBFD. Almost
136 BFDV sequences included into each genetic cluster of phylogenic tree belonged to the
137 same psittacine subfamily except for some BFDV derived from the African grey parrot.
138 Our result suggests that the possibility of cross infection among different species.
139 However, it is unclear whether these cases are specific to the African grey parrot.
140 ACKNOWLEDGMENT.
141 We thank Hiroaki Hoshino for his support and encouragement.
142 REFERENCES
143 1. Bassami, M.R., Berryman, D., Wilcox, G.E. and Raidal, S.R. 1998. Psittacine beak
144 and feather disease virus nucleotide sequence analysis and its relationship to
145 porcine circovirus, plant circoviruses, and chicken anaemia virus. Virology 249:
146 453-459.
147 2. Bassami, M.R., Ypelaar, I., Berryman, D., and Wilcox, G. E. and Raidal, S.R.
148 2001. Genetic diversity of beak and feather disease virus detected in psittacine
149 species in Australia. Virology 279: 392-400.
150 3. Bert, E., Tomassone, L., Peccati, C., Navarrete, M.G. and Sola, S.C. 2005. J. Vet.
151 Med. B Infect. Dis. Vet. Public Health 52: 64-68.
152 4. Collar, N.J. 1997. Family Psittacidae (Parrots). pp. 280-477. In: Handbook of the
9 153 Birds of the World. Vol. 4, Sandgrouse to cuckoos. (del Hoyo J., Elliott, A. and
154 Sargatal, J. eds), Lynx Edicions, Barcelona.
155 5. de Kloet, E. and de Kloet, S.R. 2004. Analysis of the beak and feather disease viral
156 genome indicates the existence of several genotypes which have a complex
157 psittacine host specificity. Arch. Virol. 149: 2393-2412.
158 6. Heath, L., Martin, D.P., Warburton, L., Perrin, M. and Horsfield, W., Kingsley, C.,
159 Rybicki, E. P. and Williamson, A.L. 2004. Evidence of unique genotypes of beak
160 and feather disease virus in southern Africa. J. Virol. 78: 9277-9284.
161 7. Hsu, C. M., Ko, C. Y. and Tsaia, H. J. 2006. Detection and sequence analysis of
162 avian polyomavirus and psittacine beak and feather disease virus from psittacine
163 birds in Taiwan. Avian Dis. 50: 348-353.5.
164 8. Kondiah, K., Albertyn, J. and Bragg, R.R. 2006. Genetic diversity of the Rep gene
165 of beak and feather disease virus in South Africa. Arch. Virol. 151: 2539-2545.
166 9. Niagro, F.D., Forsthoefel, A.N., Lawther, R.P., Kamalanathan, L., Ritchie, B.W.,
167 Latimer, K.S. and Lukert, P.D. 1998. Beak and feather disease virus and porcine
168 circovirus genomes: intermediates between the geminiviruses and plant
169 circoviruses. Arch. Virol. 143: 1723-1744.
170 10. Ogawa, H., Chahota, R., Hagino, T., Ohya, K., Yamaguchi, T. and Fukushi, H.
10 171 2006. A survey of avian polyomavirus (APV) infection in imported and domestic
172 bred psittacine birds in Japan. J. Vet. Med. Sci. 68: 743-745.
173 11. Ogawa, H., Yamaguchi, T. and Fukushi, H. 2005. Duplex shuttle PCR for
174 differential diagnosis of budgerigar fledgling disease and psittacine beak and
175 feather disease. Microbiol. Immunol. 49: 227-237.
176 12. Rahaus, M. and Wolff, M.H. 2003. Psittacine beak and feather disease: a first
177 survey of the distribution of beak and feather disease virus inside the population of
178 captive psittacine birds in Germany. J. Vet. Med. B Infect. Dis. Vet. Public Health
179 50: 368-371.
180 13. Raue, R., Johne, R., Crosta, L., Bürkle, M., Gerlach, H. and Müller, H. 2004.
181 Nucleotide sequence analysis of a C1 gene fragment of psittacine beak and feather
182 disease virus amplified by real-time polymerase chain reaction indicates a possible
183 existence of genotypes. Avian Pathol. 33: 41-50.
184 14. Ritchie, B.W. 1995. Circoviridae. pp. 223-252. In: Avian Viruses: Function and
185 Control ( Ritchie, B.W. ed.), Wingers Publishing, Florida.
186 15. Ritchie, P.A., Anderson, I.L. and Lambert, D.M. 2003. Evidence for specificity of
187 psittacine beak and feather disease viruses among avian hosts. Virology 306:
188 109-115.
11 189 16. Rowley, I. 1997. Family Cacatuidae (Cockatoos). pp.246-279. In: Handbook of the
190 Birds of the World. Vol. 4. Sandgrouse to cuckoos. (del Hoyo J., Elliott, A. and
191 Sargatal, J. eds), Lynx Edicions, Barcelona.
192 17. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. and Higgins, D. G.
193 1997. The CLUSTAL_X windows interface: flexible strategies for multiple
194 sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25:
195 4876-4882.
196 18. Ypelaar, I., Bassami, M. R., Wilcox, G.E. and Raidal, S.R. 1999. A universal
197 polymerase chain reaction for the detection of psittacine beak and feather disease
198 virus. Vet. Microbiol. 68: 141-148.
199
12 200 Figure legend
201 Fig. 1. Phylogenic tree of nucleotide sequences using the maximum-likelihood. Nucleotide
202 sequences were conducted using DNAML. Bold font indicates sequences from the present
203 study. Clear rectangles and rectangles shaded with grey indicate BFDV derived from the
204 birds of Cacatuinae and both African grey parrots and timneh grey parrot, respectively.
205 The remaining characters (denoted only by italicized font) indicate BFDV derived from
206 the birds of Psittacinae birds excluding the African grey parrot and timneh grey parrot.
207 The host bird for BFDV-USA is unknown.
13 1 Table 1. Genus of birds examined in the present study and results of BFDV examination 2 Family Genus BFDV Positive/Examined Countries and numbers 3 Subfamily (Positive rate) (Number of birds) 4 Tribe 5 Cacatuidae
6 Cacatuinae Cacatua 44/81 (51.2%) Indonesia (32/33)a, Japan (1), Singapore (10/36)a, South Africa (9), unknown (2/2)a
7 Eolophus 3/3 (100%) Indonesia (3/3)a
8 Nymphicinae Nymphicus 0/18 (0%) Japan (18)
9
10 Psittacidae
11 Lorinae Lorius 0/7 (0%) Japan (7)
12 Pseudeos 1/6 (16.7%) Singapore (1/6)a
13 Trichoglossus 2/2 (100%) unknown (2/2)a
14 Psittacinae
15 Arini Ara 1/2 (50%) U.S.A. (1/1)a, unknown (1)
16 Aratinga 2/22 (9.1%) South Africa (1/15)a, unknown (1/7)a
17 Brotogeris 0/1 (0%) Guyana (1)
18 Diopsittaca 0/2 (0%) South Africa (2)
19 Guarouba 0/2 (0%) Japan (2)
20 Pionites 0/8 (0%) Europe (4), Japan (1), Singapore (1), U.S.A. (2) 21 Pionus 2/7 (28.6%) Guyana (1), Japan (3), South Africa (2/3)a
22 Propyrrhura 0/1 (0%) Japan (1)
23 Pyrrhura 0/5 (0%) Japan (3), South Africa (2)
24 Psittacini Poicephalus 46/96(47.9%) South Africa (45/91)a, unknown (1/5)a
25 Psittacus 22/122 (18%) South Africa (22/122)a
26 Psittaculini Eclectus 1/5 (20%) Japan (1/1)a, Singapore (4)
27 Polytelis 0/3 (0%) Japan (3)
28 Psittacula 2/9 (22.2%) Japan (2), Singapore (1), South Africa (1/3)a, unknown (1/3)a
29
30 Total 126/402(31.3%) Europe (4), Guyana (2), Indonesia (35/36)a, Japan (1/42)a, Singapore (11/48)a,
31 South Africa (71/247)a, U.S.A. (1/3)a, unknown (7/20)a
3 a2 The numbers in the parenthesis indicate positive case / total number.
2 1 Table 2. Countries and results of BFDV examined
2 Countries All Birds BFDV positive (rate)
3 Europe 4 0 (0%)
4 Guyana 2 0 (0%)
5 Indonesia 36 35 (97.2%)
6 Japan 42 1 (2.4%)
7 Singapore 48 11 (22.9%)
8 South Africa 247 71 (28.7%)
9 U.S.A. 3 1 (33.3%)
10 Unknown 20 7 (35.0%)
11 Total 402 126 (31.3%)
12
1 1 Table 3. PBFDV in the present study and references used for sequence analysis
2 Code Tribe Subfamily Host Origin Accession no. References
3 AGP-SA1P-1 Psittacini Psittacinae Psittacus erithacus erithacusa South Africa AB277726 This study
4 AGP-SA1P-2 Psittacini Psittacinae Psittacus erithacus erithacusa South Africa AB277727 This study a 5 AGP-SA1P-3 Psittacini Psittacinae Psittacus erithacus erithacus South Africa AB277728 This study
6 AGP-SA1P-4 Psittacini Psittacinae Psittacus erithacus erithacusa South Africa AB277729 This study
7 AGP-SA1P-5 Psittacini Psittacinae Psittacus erithacus erithacusa South Africa AB277730 This study
8 AGP-SA2P Psittacini Psittacinae Psittacus erithacus erithacus South Africa AB277731 This study
9 TGP-SA1P Psittacini Psittacinae Psittacus erithacus timneh South Africa AB277732 This study
10 EC-SG1P Cacatuinae Cacatua galerita eleonora Singapore AB277733 This study
11 WC-SG1P Cacatuinae Cacatua alba Singapore AB277734 This study
12 WC-IN1P Cacatuinae Cacatua alba Indonesia AB277735 This study
13 YCC-IN3P Cacatuinae Cacatua sulphurea Indonesia AB277736 This study
14 YCC-IN4P Cacatuinae Cacatua sulphurea Indonesia AB277737 This study
15 BWP-SA1P Arini Psittacinae Pionus chalcopterus South Africa AB277738 This study
16 BP-SA1P Psittacini Psittacinae Poicephalus meyeri South Africa AB277739 This study
17 BHP-SA1P Psittacini Psittacinae Poicephalus cryptoxanthus South Africa AB277740 This study
18 RBP-SA1P Psittacini Psittacinae Poicephalus rufiventris South Africa AB277741 This study
19 SP-SA1P Psittacini Psittacinae Poicephalus senegalus South Africa AB277742 This study
1 20 AP-UN1P Psittaculini Psittacinae Psittacula eupatria Unknown AB277743 This study
21 EP-JP1P Psittaculini Psittacinae Eclectus roratus Japan AB277744 This study
22 DP-SA1P Psittaculini Psittacinae Psittacula derbiana South Africa AB277745 This study
23 YCC-IN1P Cacatuinae Cacatua sulphurea Indonesia AB182568 Ogawa et al. [11]
24 YCC-IN2P Cacatuinae Cacatua sulphurea Indonesia AB182569 Ogawa et al. [11]
25 RRP-JP11P Psittaculini Psittacinae Psittacula krameri Japan AB182570 Ogawa et al. [11]
26 RRP-JP12P Psittaculini Psittacinae Psittacula krameri Japan AB182571 Ogawa et al. [11]
27 BTP-SA11P Arini Psittacinae Aratinga pertinax South Africa AB182572 Ogawa et al. [11]
28 RL-JP1P Lorinae Eos bornea Japan AB182573 Ogawa et al. [11]
29 BB-WA Platycercini Psittacinae Psephotus haematogaster Australia AB182573 Bassami et al. [2]
30 LK-VIC Lorinae Trichoglossus haematodus Australia AF311299 Bassami et al. [2]
31 MMC-WA Cacatuinae Cacatua leabeateri Australia AF311300 Bassami et al. [2]
32 SCC1-WA Cacatuinae Cacatua galerita Australia AF311302 Bassami et al. [2]
33 SCC-NT Cacatuinae Cacatua galerita Australia AF311301 Bassami et al. [2]
34 LB-WA Psittaculini Psittacinae Agapornis roseicollis Australia AF311296 Bassami et al. [2]
35 Galah-WA Cacatuinae Eolophus roseicapillus Australia AF311298 Bassami et al. [2]
36 ELBC-SA Cacatuinae Cacatua tenuirostris Australia AF311297 Bassami et al. [2]
37 BFDV-AUS Cacatuinae Cacatua galerita Australia AF080560 Bassami et al. [1]
38 BFDV-USA Unknown psittacine species U. S. A. AF071878 Niagro et al. [9]
2 39 AR02-1UK Psittaculini Psittacinae Agapornis roseicollis U. K. AY521235 de Kloet et al. [5]
40 PEG07-1GE a,c Psittacini Psittacinae Psittacus erithacus erithacus Germany AY521237 de Kloet et al. [5]
41 PEU01-1UK Psittacini Psittacinae Psittacus erithacus erithacus U. K. AY521238 de Kloet et al. [5]
42 PEP01-1POR b Psittacini Psittacinae Psittacus erithacus erithacus Portugal AY521236 de Kloet et al. [5]
43 PK1-01TX Psittaculini Psittacinae Psittacula krameri U. S. A. AY521234 de Kloet et al. [5]
44 AFG3-ZA Psittacini Psittacinae Psittacus erithacus erithacus South Africa AY450443 Heath et al. [6]
45 AFG4-ZA Psittacini Psittacinae Psittacus erithacus erithacus South Africa AY450434 Heath et al. [6]
46 UC1-ZA Cacatuinae Cacatua alba South Africa AY450436 Heath et al. [6]
47 GJP1-ZA Psittacini Psittacinae Poicephalus gulielmi massaicus South Africa AY450441 Heath et al. [6]
48 RP1-ZA Psittacini Psittacinae Poicephalus rueppellii South Africa AY450439 Heath et al. [6]
49 CPA8-ZA Psittacini Psittacinae Poicephalus robustus South Africa AY450437 Heath et al. [6]
50 CPA7-ZA Psittacini Psittacinae Poicephalus robustus South Africa AY450438 Heath et al. [6]
51 BCL1-ZAM Psittaculini Psittacinae Agapornis nigrigenis Zambia AY450442 Heath et al. [6]
52 a Same individual
3 Fig. 1
BFDV-USA Unknown psittacine species U.S.A. UC1-ZA Cacatua alba , South Africa Ⅰ AFG4-ZA Psittacus erithacus erithacus, South Africa Galah-WA Eolophus roseicapillus , Australia ELBC-SA Cacatua tenuirostris , Australia AGP-SA2P Psittacus erithacus erithacus, South Africa Ⅱ SCC1-WA Cacatua galerita , Australia BFDV-AUS Cacatua galerita , Australia YCC-IN3P Cacatua sulphurea , Indonesia YCC-IN4P Cacatua sulphurea , Indonesia WC-IN1P Cacatua alba , Indonesia Ⅲ YCC-IN1P Cacatua sulphurea , Indonesia YCC-IN2P Cacatua sulphurea , Indonesia MMC-WA Cacatua leadbeateri , Australia Ⅳ SCC-NT Cacatua galerita , Australia AGP-SA1P-3 Psittacus erithacus erithacus, South Africa WC-SG1P Cacatua alba , Singapore Ⅴ EC-SG1P Cacatua galerita eleonora , Singapore RL-JP1P Eos bornea, Japan Ⅵ LK-VIC Trichoglossus haematodus, Australia Ⅶ AP-UN1P Psittacula eupatria, Unknown DP-SA1P Psittacula derbiana, South Africa BWP-SA1P Pionus chalcopterus, South Africa EP-JP1P Eclectus roratus, Japan AGP-SA1P-5 Psittacus erithacus erithacus, South Africa AGP-SA1P-1 Psittacus erithacus erithacus, South Africa Ⅷ RP1-ZA Poicephalus rueppellii, South Africa GJP1-ZA Poicephalus gulielmi massaicus, South Africa CPA7-ZA Poicephalus robustus, South Africa CPA8-ZA Poicephalus robustus, South Africa BCL1-ZAM Agapornis nigrigenis, Zambia Ⅸ RRP-JP11P Psittacula krameri, Japan Ⅹ RRP-JP12P Psittacula krameri, Japan PK1-01TX Psittacula krameri, U.S.A. AR02-1UK Agapornis roseicollis, United Kingdom ⅩⅠ BB-WA Psephotus haematogaster, Australia LB-WA Agapornis roseicollis, Australia BTP-SA11P Aratinga pertinax, South Africa ⅩⅡ RBP-SA1P Poicephalus rufiventris, South Africa ⅩⅢ PEP01-PORb Psittacus erithacus erithacus, Portugal TGP-SA1P Psittacus erithacus timneh, South Africa ⅩⅣ AFG3-ZA Psittacus erithacus erithacus, South Africa PEG07-1GE a,c Psittacus erithacus erithacus, Germany ⅩⅤ PEU01-1UK Psittacus erithacus erithacus, United Kingdom BP-SA1P Poicephalus meyeri, South Africa BHP-SA1P Poicephalus cryptoxanthus, South Africa ⅩⅥ AGP-SA1P-2 Psittacus erithacus erithacus, South Africa AGP-SA1P-4 Psittacus erithacus erithacus, South Africa SP-SA1P Poicephalus senegalus, South Africa ⅩⅦ
0.02