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 and genotype of beak and feather disease virus

2 suggesting possible interspecies cross infection during 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 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 .

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 . 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 was closely related to the

39 BFDV derived from 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 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 ( 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 ( 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 . 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 . Host birds of clusters VI and VII were Lorinae. All host birds of cluster VIII

114 to XVII were . 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

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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.,

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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

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10 171 2006. A survey of avian polyomavirus (APV) infection in imported and domestic

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173 11. Ogawa, H., Yamaguchi, T. and Fukushi, H. 2005. Duplex shuttle PCR for

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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

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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 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 -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