Avian Pox in Native Captive Psittacines, Brazil, 2015

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Technical Appendix

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Abstract

To investigate an outbreak of avian pox in psittacines in a conservation facility, we examined 94 birds of 10 psittacine species, including sick and healthy birds. We found psittacine pox virus in 23 of 27 sick birds and 4 of 67 healthy birds. Further characterization is needed for these isolates.

Avian pox is caused by avipoxvirus. Infections occur worldwide in domestic and wild avian species (1), are suggested to be host family- or order-specific, and are modulated by habitat and ecologic niche (2). Avipoxviruses have been described in Brazilian Amazona spp. and Pionus spp. parrots with severe diphtheritic upper digestive lesions, experimentally causing the formation of cutaneous lesions in chickens; chicken and parrot strains will not provide cross protection (3). The presumptive diagnosis, based on typical pocklike skin lesions of papular or nodular hyperplasic and hypertrophic skin foci or upper digestive diphtheritic form in severe cases (1), may be confirmed by detection of avipoxvirus DNA by PCR (4).

In June 2015, an outbreak of avian pox occurred among 10 species of native Brazilian psittacines (n = 94) maintained in a conservation facility. In addition to the typical pocklike nodular skin lesions, the psittacines had weight loss and reduced activity; 3 died. The outbreak lasted 3 months; remission of lesions occurred within ≈3 weeks in each bird.

Skin scrapings were collected from the cutaneous lesions of affected birds (Technical Appendix[PDF - 378 KB - 4 pages]Figure 1), and conjunctiva and cloaca swabs were collected from all 94 psittacines showing cutaneous lesions (27 birds) or not (67 birds). Skin samples treated with an antibacterial–antimycotic drug mixture (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) were inoculated onto the chorioallantoic membrane (CAM) of 10-day-old specific pathogen–free chicken embryos and typical pocklike CAM lesions were obtained (Technical Appendix[PDF - 378 KB - 4 pages]Figure 2). Cutaneous samples and the CAM of inoculated embryos were subjected to PCR with specific primers forward 5′-CAGCAGGTGCTAAACAACAA-3′ and reverse 5′-CGGTAGCTTAACGCCGAATA-3′ (4) to amplify a partial sequence (576 bp) of the gene encoding the core protein P4b (fpv167 locus) of avipoxvirus (Table). The avipoxvirus P4b gene partial sequence was obtained from skin lesions, conjunctiva and cloacal swabs, and CAM; sequences then underwent phylogenetic characterization (Technical Appendix[PDF - 378 KB - 4 pages]Figure 3).

The P4b gene partial sequences obtained from the avipoxvirus isolate Betim-1 of the red-and-green macaw (Ara chloropterus) (GenBank accession no. KT187552), dusky parrot (Pionus fuscus) (Esteves et al., unpub. data), and golden conure (Guaruba guarouba) (Esteves et al., unpub. data) were 100% identical. The obtained sequences were aligned with local avipoxvirus of different species with clinical disease (Esteves et al., unpub. data) and with previously published avipoxvirus sequences in GenBank (Technical Appendix[PDF - 378 KB - 4 pages]Figure 3). Evolutionary analyses were performed in MEGA6 (5), evolutionary history was inferred by neighbor joining (6), replicate trees were clustered by using the bootstrap test (7) with 1,000 oversampling, and evolutionary distances were computed by the Tamura 3-parameter method (8). The phylogenetic relationships revealed grouping with psittacine poxviruses, including isolates from the United Kingdom (macaw, GenBank accession no. AM050382.1, and parrot, GenBank accession no. AM050383.1); United States (yellow-crowned amazon [Amazona ochrocephala], GenBank accession no. KC018069.1); and Germany (lovebird [Agapornissp.], GenBank accession no. AY530311.1). To enable comparisons, a local strain (GenBank accession no. KX863707) of the Atlantic canary (Serinus canaria) (Esteves et al., unpub. data) and an isolate of the Magellanic penguin (Spheniscus magellanicus) (GenBank accession no. KF516679.1) (9) were grouped in the canarypox clade. A local chicken isolate (GenBank accession no. KX863706) was grouped within the fowlpox clade, including chicken (GenBank accession nos. KF722860.1 KF722863.1) and turkey (GenBank accession nos. KF017961.1 and DQ873808.1) strains.

Birds with pocklike lesions represented 28.7% (27/94) of psittacines in the sanctuary. Laboratory diagnosis was implemented at the early stages of the outbreak; birds were clinically observed up to the final cases (3 months). Of the 27 psittacines with lesions, 23 (85.2%) were PCR positive for avipoxvirus. No blue-fronted parrots (Amazona aestiva) showed lesions, but all were PCR positive (4/94 [4.2%]), suggesting that they were immune, although they were not tested for an immune response. Among the 25 golden conures, the most abundant species in the premises, 9 showed typical pocklike lesions and were PCR positive, but 1 with lesions was PCR negative and 15 had no lesions and were PCR negative, indicating nonuniform immunity. None of the 14 blue and yellow macaws (Ara ararauna) had lesions; PCR results were negative for all, suggesting previous immunity or a lower susceptibility to infection. Four birds with lesions were not PCR positive, possibly indicating an advanced phase of virus-free scars.

The proximity of aviaries suggests that all birds might have had the opportunity for infection during the outbreak. The death rate (3.2%) for the outbreak was low; the death of young golden conures with diphtheritic lesions and 2 adult dusky parrots that demonstrated conjunctivitis and cachexia were accompanied by complications by Candida albicans and Capillaria sp., suggesting the potential aggravation risk. Most psittacines (63/94; 67.0%) were PCR negative and without lesions, as tested in cloacal and conjunctival swabs.

The affected species are declining in wild populations. Our findings emphasize the risk for avipoxvirus among captive psittacines; their relevance for psittacine rehabilitation and conservation may be considerable regarding pathogenicity. As shown, 2 adult dusky parrots and 1 nestling golden conure died; further characterization is needed for the isolates, including their eventual importance for commercial poultry.

Dr. Esteves is a veterinarian dedicated to native wildlife conservation. He is the leader of a nongovernmental agency dedicated to the rescue and rehabilitation of wild animals. On his farm, he maintains and cares for about 170 animals that are not fit to reintroduce into nature.

Felipe C.B. Esteves, Sandra Y. Marín, Maurício Resende, Aila S.G. Silva, Hannah L.G. Coelho, Mayara B. Barbosa, Natália S. D’Aparecida, José S. de Resende, Ana C.D. Torres, and Nelson R.S. Martins

Author affiliations: Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

Acknowledgment

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvovimento Científico e Tecnológico (CNPq), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) provided financial support and scholarships.

References

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Table

Table. Avipoxvirus detection by PCR according to clinical status and psittacine species, Brazil, 2015

Technical Appendix

Technical Appendix. Green-winged macaw with facial lesions, virus isolation in the chorioallantoic membrane with pocklike lesions, and the phylogenetic tree showing grouping of an isolate with Psittacinepoxvirus. 378 KB

Suggested citation for this article: Esteves FCB, Marín SY, Resende M, Silva ASG, Coelho HLG, Barbosa MB, et al. Avian pox in native captive psittacines, Brazil, 2015. Emerg Infect Dis. 2017 Jan [date cited]. http://dx.doi.org/10.3201/eid2301.161133

DOI: 10.3201/eid2301.161133