Psittacine Beak and Feather Disease–like Illness in Gouldian Finches (Chloebia gouldiae) Author(s): Elena Circella, Marilisa Legretto, Nicola Pugliese, Anna Caroli, Giancarlo Bozzo, Gianluca Accogli, Antonio Lavazza, and Antonio Camarda Source: Avian Diseases, 58(3):482-487. 2014. Published By: American Association of Avian Pathologists DOI: http://dx.doi.org/10.1637/10745-121113Case.1 URL: http://www.bioone.org/doi/full/10.1637/10745-121113Case.1
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AVIAN DISEASES 58:482–487, 2014
Case Report— Psittacine Beak and Feather Disease–like Illness in Gouldian Finches (Chloebia gouldiae) Elena Circella,AD Marilisa Legretto,A Nicola Pugliese,A Anna Caroli,A Giancarlo Bozzo,A Gianluca Accogli,B Antonio Lavazza,C and Antonio CamardaA A Department of Veterinary Medicine, University of Bari ‘‘Aldo Moro’’, S. P. Casamassima km. 3, 70010, Valenzano, Bari, Italy Department of Emergency and Organ Transplantation, University of Bari ‘‘Aldo Moro’’, S. P. Casamassima km. 3, 70010, Valenzano, Bari, Italy C Virology Department, Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna ‘‘Bruno Ubertini’’, Via Antonio Bianchi 7/9, 25124, Brescia, Italy B
Received 20 December 2013; Accepted 26 February 2014; Published ahead of print 27 February 2014 SUMMARY. Beak and feather disease virus (BFDV) is a member of the genus Circovirus and causes psittacine beak and feather disease (PBFD) in Psittaciformes. PBFD is a severe disease generally characterized by immunodeficiency and beak and feather disorders. Although Circovirus spp. have been detected in several nonpsittacine species, little is known about the symptoms and the disease associated with this infection in birds other than Psittaciformes. In this study, we report the identification of Circovirus infection in a flock of Gouldian finches showing beak and feather disorders. Sequence analyses on the rep gene of the virus highlighted a strong similarity at nucleotide and amino acid levels with the corresponding regions of BFDV from psittacine species. By contrast, it was more distant to circoviruses identified in finch and canary. RESUMEN. Reporte de Caso—Problema similar a la enfermedad del pico y las plumas (PBFD) de los psita´cidos en diamantes de Gould (Chloebia gouldiae). El virus de la enfermedad del pico y de las plumas (BFDV) es un miembro del ge´nero Circovirus y causa la enfermedad del mismo nombre (con las siglas en ingle´s PBFD) en psita´cidos. La enfermedad del pico y las plumas es una enfermedad grave caracterizada generalmente por inmunodeficiencia y trastornos del pico y las plumas. Aunque se han detectado Circovirus spp. en varias especies no psita´cidas, poco se sabe acerca de los signos y enfermedad asociadas con esta infeccio´n en las aves distintas a las Psittaciformes. En este estudio, se presenta la identificacio´n de la infeccio´n por circovirus en una parvada de pinzones de Gould que mostraron trastornos de pico y plumas. El ana´lisis de la secuencia del gene rep del virus mostro´ una gran similitud a nivel de nucleo´tidos y de aminoa´cidos con las regiones correspondientes al virus de la enfermedad del pico y plumas de las especies de psita´cidos. Por el contrario, este virus fue ma´s distante al circovirus identificado en el pinzo´n y en el canario. Key words: Circovirus, Gouldian finch, PBFD, beak and feather Abbreviations: APV 5 avian polyoma virus; BFDV 5 beak and feather disease virus; CaCV 5 canary circovirus; CoCV 5 columbid circovirus; CP 5 coat protein; EM 5 electron microscopy; FiCV 5 finch circovirus; H&E 5 hematoxylin and eosin; NaPT 5 sodium phosphotungstic acid; PBFD 5 psittacine beak and feather disease; PCV 5 porcine circovirus; rep 5 replicationassociated protein; TEM 5 transmission electron microscopy; TN 5 Tamura–Nei
Members of genus Circovirus, family Circoviridae, are nonenveloped viruses that possess a circular single-stranded DNA genome about 2 kb in size (22). The genome encodes a replicationassociated protein (rep) and a coat protein (CP). Another open reading frame has been detected, but its role is still undefined (37). Beak and feather disease virus (BFDV) belongs to genus Circovirus, and it is responsible for psittacine beak and feather disease (PBFD). PBFD is characterized by feather and beak disorders and immunodeficiency. Feather disorders may occur in the acute or chronic course of PBFD, and they consist of bending, fractures, bleeding, premature shedding of feather, retention of feather sheaths, and necrosis (7). Beak disorders occur particularly in the chronic course of the infection. They consist of changes of color and shape, progressive elongation, fractures, and necrosis and can be so severe and painful as to suggest euthanasia (7). The immunodeficiency is due to the depletion of lymphoid tissues, such as the bursa of Fabricius and the thymus, often predisposing the affected birds to secondary bacterial or fungal infections (13,32). D
Corresponding author: E-mail: [email protected]
Currently, BFDV infection is considered a worldwide infection in psittacine species and has been detected in more than 60 different species (2,32). Members of genus Circovirus also have been detected in nonpsittacine birds, such as canaries (26,35), pigeons (5,18,33), ostriches (6,28), geese (3,36), and ducks (29). The clinical course and the signs associated with the infection are not yet well known and defined in those species. Circovirus has also been detected in Gouldian finch (27), but it has never been related to beak and feather disease. The infection was associated with respiratory symptoms and to severe lymphoid depletion, particularly in the bursa of Fabricius of the affected birds. That virus was classified as a new member of the genus Circovirus (34). In this paper, a Circovirus infection associated with beak and feather disorders in a flock of Gouldian finches is reported.
MATERIALS AND METHODS Case history. The flock consisted of five pairs of breeders (from 2 to 3 yr of age) and six young finches (about 3 mo of age). No other bird species, such as the society finch (Bengalese finch, Lonchura striata
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Fig. 1. Beak and feather disorders observed in the infected flock. (A, B) Beak lesions. (C, D) Feather loss and beak lesions. The presence of feather sheaths is due to the molt process. domestica), were present. Usually, society finches are used to foster baby Gouldians. On the contrary, Gouldian finch breeders of the examined flock were perfectly adapted to the aviary environment, and they easily took care of their chicks. The finches were reared outside in a single aviary (dimensions: 2 m 3 2 m 3 2 m). All birds were moved indoors into a smaller cage (dimensions 80 cm 3 70 cm 3 1.20 cm) at the end of each reproduction season during the colder months of the year. Five adults (three males and two females) progressively developed deformities of the beak, which appeared elongated and rough (Fig. 1). These abnormalities appeared and rapidly advanced in a few months. Among the five birds with beak anomalies, a female also showed heavy feather loss to the head and neck region. Nutritional support with vitamins and sulfur-containing amino acids was administrated without any improvement. No feather or beak disorders were observed in the young finches, but two of them showed weakness, lethargy, and ruffled plumage. Therapy with enrofloxacin (200 mg/L drinking water) was unsuccessfully administered to the flock by the owner. The sick young finches died at different times during the following weeks. Samples of feathers, scrapings from beaks, and feces from the five breeders and the two dead young finches were sent for diagnostic purposes to the Division of Avian Diseases of the Department of Veterinary Medicine, University of Bari, Italy. Bacteriologic and parasitologic investigations. Samples of liver, spleen, cardiac blood, and gut were collected from the two dead birds at postmortem examination. Bacteriologic investigations were performed using selective (MacConkey agar, Oxoid, Milan, Italy) and nonselective (blood agar, Oxoid) media in aerobic and anaerobic conditions at 37 C for 24 hr. Fresh smears were prepared from the fecal samples from the
five affected breeders and from the intestinal content of the two dead birds for direct microscopic examination. Each sample was also observed after flotation in saturated NaCl solution with 650 g/L glucose. Histology. Samples of liver were obtained during postmortem examination of the two young finches. For histologic analysis, the blocks of tissue were collected and fixed in 10% neutral buffered formalin. The samples were embedded in paraffin wax, sectioned at 4 mm and stained by hematoxylin and eosin (H&E). Virology. Feather quills were cut and mechanically homogenized in a few drops of distilled water for negative-staining electron microscopy (EM) examination. The method used (15) is based on concentration of viral particles through Airfuge (Beckman Coulter Inc., Brea, CA) ultracentrifugation. Grids were stained with 2% sodium phosphotungstic (NaPT), pH 6.8, for 1.5 min and examined at 19–30000 with a Tecnai G2 Spirit transmission electron microscope (TEM; FEI, Eindhoven, The Netherlands) operating at 85 kV. Viral particles were identified based on their morphologic characteristics. Samples of liver, spleen, gut, and feather from the two finches examined postmortem were screened by PCR to evaluate infections by members of genus Circovirus and avian polyoma virus (APV). Moreover, a PCR assay was used to screen scrapings from beak, feathers, and feces from adults with beak and feather disorders. DNA extraction was performed from each sample by using EuroGold Tissue Mini Kit (EuroClone, Milan, Italy) according to the manufacturer’s instructions. The PCRs were carried out in a mixture containing 1 U of platinum Taq polymerase (Invitrogen, Milan, Italy), 20 mM Tris–HCl, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, and 25 pmol of each primer. Up to 400 ng of total DNA from sample tissues were added to the mixture. Primers DCiVf/DCiVr (28) were used for Circovirus detection.
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Table 1. Comparison of the sequenced rep gene fragment with the corresponding portion of representative members of genus Circovirus. GenBank accession numbers and BFDV hosts are reported.A TN+G distance from % Nucleotide identity EC32/ITA/2011 with EC32/ITA/2011
CaCV AJ301633 FiCV DQ845075 PCV1 U49186 PCV2 AF055392 CoCV AF252610 BFDV1 AF311299 Tha BFDV2 FJ685979 Csu BFDV3 EF457975 Nho BFDV4 AY521238 Per BFDV AY521235 Aro
0.564 0.535 1.056 1.056 0.532 0.102 0.049 0.073 0.048 0.053
63.13 64.53 48.50 50.30 64.53 90.78 95.39 93.19 95.39 94.99
A Tha 5 Trichoglossus haematodus; Csu 5 Cacatua sulphurea; Nho 5 Nymphicus hollandicus; Per 5 Psittacus erithacus; Aro 5 Agapornis roseicollis.
A touchdown PCR protocol was set up due to the high difference between melting temperatures of primer DCiVf and DCiVr. Cycling conditions were as follow: a 5 min denaturation step at 94 C; 14 cycles performed, each with 30 sec of denaturation at 94 C; 30 sec of annealing at 55 C for the first cycle, decreasing by 0.5 C for each of the next cycles; and 1 min of extension at 72 C. A further 21 cycles were conducted as follows: 94 C for 30 sec, 49 C for 30 sec, and 72 C for 1 min, with a final extension at 72 C for 10 min. Primers and cycling conditions previously described (17) were used for APV detection. The PCR products were visualized after agarose gel electrophoresis and 0.5 mg/ml ethidium bromide staining. Cloning and sequence analysis. Two PCR amplicons generated from the liver of a young finch and from the beak of a breeder finch were selected, purified, cloned, and sequenced. The PCR products were purified using the EuroGold Cycle-Pure Kit (EuroClone). Each purified product was cloned in pGEMH-T Easy cloning vector (Promega, Milan, Italy), according to the manufacturer’s protocol. Three to five positive clones were selected for each cloning experiment, and their inserts were sequenced using the Big Dye Terminator v3.1. Sequence determination was performed on an Applied Biosystem ABI 3100 at the facilities of Bmr Genomics (Padua, Italy). The nucleotide sequences from each cloned sample were assembled by using the SeqManII program (DNAStar, Madison, WI). Identification of the product was carried out by comparison with the sequences in GenBank by BLAST. Alignment between the two sequences was performed by ClustalW algorithm (gap open penalty 5 10, gap extension penalty 5 5) implemented in MEGA5 (30). Because the sequences were 100% identical to each other, only one of them was selected and aligned with the corresponding fragment of the rep gene from a representative panel of porcine circovirus (PCV), finch circovirus (FiCV), canary circovirus (CaCV), columbid circovirus (CoCV), and BFDV. The BFDV strains were selected to represent strains identified in each family of the Psittaciformes order. The sequences are listed in Table 1. Alignment was performed by ClustalW with the above-mentioned parameter and with manual editing. The best model for phylogenetic analyses was determined by using Modeltest (24) implemented in FindModel (available online at http://www.hiv.lanl.gov/content/ sequence/findmodel/findmodel.html). The selected model was Tamura– Nei (TN)+G, with gamma distribution shape (a 5 0.49). MEGA was also used to calculate pairwise and group nucleotide and phylogenetic (according to the TN model) distances among sequences. Maximum likelihood phylogenies of the BFDV genomes were inferred using PHYML (10) with 1000 nonparametric bootstrap replicates. The sequence of the analyzed rep gene fragment has been submitted to GenBank with the accession number JX131620.
Fig. 2. Light microscopy images of a liver section. Intranuclear Cowdry type B inclusion bodies (In) and necrosis area (N). H&E 10003 magnification. RESULTS
At postmortem examination, the birds appeared dehydrated and underweight. The liver appeared congested and enlarged. No other gross lesions were evident. Specific bacterial and parasitic pathogens were not detected, as all tests gave negative results. The histologic sections of the livers showed intranuclear Cowdry type B inclusion bodies and chromatin marginalization. Necrotic cells scattered throughout the liver parenchyma were also observed (Fig. 2). Electron microscopy observation of feather quill homogenates revealed the presence of few scattered, small (18–20 nm) icosahedral viral particles without envelope, morphologically related to circovirus (Fig. 3).
Fig. 3. Ultramicrograph of virions observed in the feather quill extract. Icosahedral particles of about 18–20 nm diameter morphologically resembling circovirus. Negative staining 2% NaPT, pH 6.8. FEI Tecnai G2 Spirit TEM at 85 kV. Bar 5 100 nm.
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DISCUSSION
Fig. 4. Maximum likelihood tree of the analyzed rep gene fragment. Internal labels contain the species abbreviation, the GenBank accession number, and, for BFDV, the host abbreviations. Significant bootstrap values are indicated for the main nodes. Tha 5 Trichoglossus haematodus; Csu 5 Cacatua sulphurea; Nho 5 Nymphicus hollandicus; Per 5 Psittacus erithacus; Aro 5 Agapornis roseicollis.
PCR for APV was always negative. On the contrary, PCR for the Circovirus genus generated an amplicon of the expected size (550 bp) from the two liver samples and from all the tested samples from young finches and breeders. Circovirus sequence analysis. Two amplicons were cloned and sequenced. The resultant sequences were identical to each other. Upon BLAST analysis, the best match (97%) was found for the correspondent sequence of the rep gene of a BFDV identified in an African grey parrot (Psittacus erithacus; accession EU810207). Therefore, the virus identified in this study, strain EC32/ITA/ 2011, was characterized as Circovirus. To assess the phylogenetic relationships of the rep gene sequence of EC32/ITA/2011 with other circoviruses, the sequence was aligned with a representative panel of corresponding sequences from BFDV, CaCV, CoCV, FiCV, and PCV retrieved in GenBank. The phylogenetic analysis revealed that EC32/ITA/2011 was closely related to BFDV, whereas it was more distant from the reported FiCV and CaCV sequences, with strong bootstrap support (Fig. 4). Pairwise distances (computed by the TN+G model) between EC32/ ITA/2011 and FiCV and CaCV were 0.535 and 0.564, respectively. By contrast, the mean pairwise distance between EC32/ITA/2011 and the sequences belonging to the BFDV group was 0.065, with nucleotide identity ranging from 91 (Trichoglossus haematodus BFDV) to 95% (P. erithacus BFDV; Table 1).
The histologic findings observed in the liver sections in the young finches, and specifically the finding of inclusion bodies, highlighted a viral infection. Since Polyomavirus, which may cause very similar lesions in the beak and liver of Estrildidae, was not detected, we hypothesize an etiologic role of Circovirus, even if it is known that it also occurs subclinically (32). Nevertheless, other pathogens, such as Macrorhabdus ornithogaster, Candida albicans, Atoxoplasma spp., and Chlamydia psittaci, should be considered in the differential diagnosis, even if they are not commonly associated with the signs and lesions observed in the outbreak. In particular, M. ornithogaster (19) and C. albicans (16) are responsible for mortality in young birds and nestlings, but symptoms, such as regurgitation and vomiting, and lesions in the upper gastrointestinal tract were not observed. Actually, the liver lesions observed at postmortem examination (i.e., enlargement and congestion) resembled those caused by Atoxoplasma spp., but we did not observed in the histologic sections the sporozoites, that are typically found in the liver (25). Chlamydia psittaci is not frequently found in finches, but it has been described recently in canaries (4). It generally causes severe hepatitis and perihepatitis and liver necrosis (1,11). Little is known about the characteristics of Circovirus infections in nonpsittacine birds, and even the associated clinical signs are not yet well defined. A member of the genus Circovirus was identified in a flock of Gouldian finches (24), and it was then considered a novel Circovirus (FiCV) (34). The infection was associated with a respiratory syndrome characterized by lethargy, nasal discharge, severe dyspnea, and death. Previously, the detection of circovirus-like particles was associated with feather loss and liver necrosis in young Zebra finches (21). In other nonpsittacine species, Circovirus was associated with a ‘‘black spot’’ condition characterized by abdominal enlargement and gall bladder congestion in neonatal canaries (8). In adult canaries, the infection was found to be associated with lethargy, anorexia, and feather loss followed by death (35). The virus identified from canaries was later considered a novel Circovirus because of its phylogenetic distance from BFDV and other avian circoviruses (23). Beak pathologies have never been associated with Circovirus infection in Gouldian finches and other nonpsittacine species, whereas they are typical of PBFD in psittacine birds (32). Although more rarely detected than feather disorders, beak abnormalities are observed in the chronic course of PBFD, and they are relatively common in some species, such as cockatoos (7,12,20). The symptoms we observed in the Gouldian finches primarily resemble PBFD rather than the clinical signs described in Circovirus infection in nonpsittacine species. In particular, feather loss was found, but the most evident signs were observed in the beaks of the adult. The lack of evident beak abnormalities in the younger finches was probably due to the different course of the disease. Because the disease progressed more rapidly in the young than in their adult counterparts, the former probably died before developing beak disorders. Moreover, sequence analyses highlighted a substantial identity between the rep gene of EC32/ITA/2011 and those of BFDV, whereas it appeared more distant from FiCV and CaCV. Considering that recent studies (14) evidenced changes and mutations, in particular in important regions of the normally conserved rep gene and not of the cp gene, the possibility of an interspecific spread of BFDV to Gouldian finches, causing the typical clinical features of PBFD, could be suggested. Gouldian finches showed a more rapid course in the development of clinical signs, in particular in the progression of beak lesions with respect to psittacine species. This relatively rapid onset may be due
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to a difference in behavior of the virus in an unusual host such as the Gouldian finch, but the data available at this time are not enough to verify such hypothesis; therefore, further investigations are currently in progress. How the Gouldian finch flock may have acquired the infection remains unclear. The owner has often come in contact with other finch and psittacine bird farmers, and he attended local bird fairs. Therefore, he may have played a role as a vehicle of the virus, considering the widely recognized high stability of BFDV in the environment (31). Nonetheless, all finches were reared outdoors for several months a year, so contact with other potential carriers of the virus cannot be excluded. Interestingly, monk parakeets (Myiopsitta monachus) live as wild birds a few kilometers from the finch aviary; they were probably previously reared as pet birds who, after escape, adapted to wild life and reproduced. Because monk parakeets have a high dispersal potential (9), some of them could have been in proximity of the Gouldian finch flock. However, the circulation of BFDV among the colony has not been verified to date. REFERENCES 1. Andersen, A. A., and D. Vanrompay. Avian chlamydiosis (psittacosis, ornithosis). In: Diseases of poultry, 12th ed. Y. M. Saif, ed. Blackwell Publishing, Ames, IA. pp. 971–986. 2008. 2. Cathedral-Ortiz, L., B. Kurenbach, M. Massaro, K. McInnes, D. H. Brunton, M. E. Hauber, D. P. Martin, and A. Varsani. A new isolate of beak and feather disease virus from endemic wild red-fronted parakeets (Cyanoramphus novaezelandiae) in New Zealand. Arch. Virol. 155:613–620. 2010. 3. Chen, C. L., P. C. Chang, M. S. Lee, J. H. Shein, S. J. Ou, and H. K. Shieh. Nucleotide sequences of goose circovirus isolated in Taiwan. Avian Pathol. 32:165–171. 2003. 4. Circella, E., N. Pugliese, G. Todisco, M. A. Cafiero, O. A. E. Sparagano, and A. Camarda. Chlamydia psittaci infection in canaries heavily infested by Dermanyssus gallinae. Exp. Appl. Acarol. 55:329–338. 2011. 5. Duchatel, J. P., D. Todd, J. A. Smyth, J. C. Bustin, and H. Vindevogel. Observations on detection, excretion and transmission of pigeon circovirus in adult, young and embryonic pigeons. Avian Pathol. 35:30–34. 2006. 6. Eisenberg, S. W. F., A. J. A. M. van Asten, A. M. van Ederen, and G. M. Dorrestein. Detection of circovirus with a polymerase chain reaction in the ostrich (Struthio camelus) on farm in the Netherlands. Vet. Microbiol. 95:27–38. 2003. 7. Gerlach, H. Circoviridae—psittacine beak and feather disease virus. In: Avian medicine: principles and application. B. W. Ritchie, J. Harrison, and L. R. Harrison, eds. Wingers Publishing, Lake Worth, FL. pp. 894–902. 1994. 8. Goldsmith, T. L. Documentation of passerine circoviral infection. In: Proc. Annual Conference of the American Association of Avian Veterinarians, Philadelphia, PA. pp. 349–350. 1995. 9. Gonc¸alves da Silva, A., J. R. Eberhard, T. F. Wright, M. L. Avery, and M. A. Russello. Genetic evidence for high propagule pressure and longdistance dispersal in monk parakeet (Myiopsitta monachus) invasive populations. Mol. Ecol. 19:3336–3350. 2011. 10. Guindon, S., and O. Gascuel. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52:696–704. 2003. 11. Harkinezhad, T., T. Geens, and D. Vanrompay. Chlamydophila psittaci infections in birds: a review with emphasis on zoonotic consequences. Vet. Microbiol. 135:68–77. 2009. 12. Jacobson, E. R., S. Clubb, C. Simpson, M. Walsh, C. D. Lothrop Jr., J. Gaskin, J. Bauer, S. Hines, G. V. Kollias, P. Poulos, and G. Harrison. Feather and beak dystrophy and necrosis in cockatoos: clinicopathologic evaluations. J. Am. Vet. Med. Assoc. 189:999–1005. 1986. 13. Katoh, H., H. Ogawa, K. Ohya, and H. Fukushi. A review of DNA viral infections in psittacine birds. J. Vet. Med. Sci. 72:1099–1106. 2010.
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37. Varsani, A., G. K. de Villiers, G. L. Regnard, R. R. Bragg, K. Kondiah, I. I. Hitzeroth, and E. P. Rybicki. A unique isolate of beak and feather disease virus isolated from budgerigars (Melopsittacus undulatus) in South Africa. Arch. Virol. 155:435–439. 2010.
ACKNOWLEDGMENT We thank Giovanni Circella of the University of California, Davis, CA, for reviewing the English in the manuscript.
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AVIAN DISEASES 58:482–487, 2014
Case Report— Psittacine Beak and Feather Disease–like Illness in Gouldian Finches (Chloebia gouldiae) Elena Circella,AD Marilisa Legretto,A Nicola Pugliese,A Anna Caroli,A Giancarlo Bozzo,A Gianluca Accogli,B Antonio Lavazza,C and Antonio CamardaA A Department of Veterinary Medicine, University of Bari ‘‘Aldo Moro’’, S. P. Casamassima km. 3, 70010, Valenzano, Bari, Italy Department of Emergency and Organ Transplantation, University of Bari ‘‘Aldo Moro’’, S. P. Casamassima km. 3, 70010, Valenzano, Bari, Italy C Virology Department, Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna ‘‘Bruno Ubertini’’, Via Antonio Bianchi 7/9, 25124, Brescia, Italy B
Received 20 December 2013; Accepted 26 February 2014; Published ahead of print 27 February 2014 SUMMARY. Beak and feather disease virus (BFDV) is a member of the genus Circovirus and causes psittacine beak and feather disease (PBFD) in Psittaciformes. PBFD is a severe disease generally characterized by immunodeficiency and beak and feather disorders. Although Circovirus spp. have been detected in several nonpsittacine species, little is known about the symptoms and the disease associated with this infection in birds other than Psittaciformes. In this study, we report the identification of Circovirus infection in a flock of Gouldian finches showing beak and feather disorders. Sequence analyses on the rep gene of the virus highlighted a strong similarity at nucleotide and amino acid levels with the corresponding regions of BFDV from psittacine species. By contrast, it was more distant to circoviruses identified in finch and canary. RESUMEN. Reporte de Caso—Problema similar a la enfermedad del pico y las plumas (PBFD) de los psita´cidos en diamantes de Gould (Chloebia gouldiae). El virus de la enfermedad del pico y de las plumas (BFDV) es un miembro del ge´nero Circovirus y causa la enfermedad del mismo nombre (con las siglas en ingle´s PBFD) en psita´cidos. La enfermedad del pico y las plumas es una enfermedad grave caracterizada generalmente por inmunodeficiencia y trastornos del pico y las plumas. Aunque se han detectado Circovirus spp. en varias especies no psita´cidas, poco se sabe acerca de los signos y enfermedad asociadas con esta infeccio´n en las aves distintas a las Psittaciformes. En este estudio, se presenta la identificacio´n de la infeccio´n por circovirus en una parvada de pinzones de Gould que mostraron trastornos de pico y plumas. El ana´lisis de la secuencia del gene rep del virus mostro´ una gran similitud a nivel de nucleo´tidos y de aminoa´cidos con las regiones correspondientes al virus de la enfermedad del pico y plumas de las especies de psita´cidos. Por el contrario, este virus fue ma´s distante al circovirus identificado en el pinzo´n y en el canario. Key words: Circovirus, Gouldian finch, PBFD, beak and feather Abbreviations: APV 5 avian polyoma virus; BFDV 5 beak and feather disease virus; CaCV 5 canary circovirus; CoCV 5 columbid circovirus; CP 5 coat protein; EM 5 electron microscopy; FiCV 5 finch circovirus; H&E 5 hematoxylin and eosin; NaPT 5 sodium phosphotungstic acid; PBFD 5 psittacine beak and feather disease; PCV 5 porcine circovirus; rep 5 replicationassociated protein; TEM 5 transmission electron microscopy; TN 5 Tamura–Nei
Members of genus Circovirus, family Circoviridae, are nonenveloped viruses that possess a circular single-stranded DNA genome about 2 kb in size (22). The genome encodes a replicationassociated protein (rep) and a coat protein (CP). Another open reading frame has been detected, but its role is still undefined (37). Beak and feather disease virus (BFDV) belongs to genus Circovirus, and it is responsible for psittacine beak and feather disease (PBFD). PBFD is characterized by feather and beak disorders and immunodeficiency. Feather disorders may occur in the acute or chronic course of PBFD, and they consist of bending, fractures, bleeding, premature shedding of feather, retention of feather sheaths, and necrosis (7). Beak disorders occur particularly in the chronic course of the infection. They consist of changes of color and shape, progressive elongation, fractures, and necrosis and can be so severe and painful as to suggest euthanasia (7). The immunodeficiency is due to the depletion of lymphoid tissues, such as the bursa of Fabricius and the thymus, often predisposing the affected birds to secondary bacterial or fungal infections (13,32). D
Corresponding author: E-mail: [email protected]
Currently, BFDV infection is considered a worldwide infection in psittacine species and has been detected in more than 60 different species (2,32). Members of genus Circovirus also have been detected in nonpsittacine birds, such as canaries (26,35), pigeons (5,18,33), ostriches (6,28), geese (3,36), and ducks (29). The clinical course and the signs associated with the infection are not yet well known and defined in those species. Circovirus has also been detected in Gouldian finch (27), but it has never been related to beak and feather disease. The infection was associated with respiratory symptoms and to severe lymphoid depletion, particularly in the bursa of Fabricius of the affected birds. That virus was classified as a new member of the genus Circovirus (34). In this paper, a Circovirus infection associated with beak and feather disorders in a flock of Gouldian finches is reported.
MATERIALS AND METHODS Case history. The flock consisted of five pairs of breeders (from 2 to 3 yr of age) and six young finches (about 3 mo of age). No other bird species, such as the society finch (Bengalese finch, Lonchura striata
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Fig. 1. Beak and feather disorders observed in the infected flock. (A, B) Beak lesions. (C, D) Feather loss and beak lesions. The presence of feather sheaths is due to the molt process. domestica), were present. Usually, society finches are used to foster baby Gouldians. On the contrary, Gouldian finch breeders of the examined flock were perfectly adapted to the aviary environment, and they easily took care of their chicks. The finches were reared outside in a single aviary (dimensions: 2 m 3 2 m 3 2 m). All birds were moved indoors into a smaller cage (dimensions 80 cm 3 70 cm 3 1.20 cm) at the end of each reproduction season during the colder months of the year. Five adults (three males and two females) progressively developed deformities of the beak, which appeared elongated and rough (Fig. 1). These abnormalities appeared and rapidly advanced in a few months. Among the five birds with beak anomalies, a female also showed heavy feather loss to the head and neck region. Nutritional support with vitamins and sulfur-containing amino acids was administrated without any improvement. No feather or beak disorders were observed in the young finches, but two of them showed weakness, lethargy, and ruffled plumage. Therapy with enrofloxacin (200 mg/L drinking water) was unsuccessfully administered to the flock by the owner. The sick young finches died at different times during the following weeks. Samples of feathers, scrapings from beaks, and feces from the five breeders and the two dead young finches were sent for diagnostic purposes to the Division of Avian Diseases of the Department of Veterinary Medicine, University of Bari, Italy. Bacteriologic and parasitologic investigations. Samples of liver, spleen, cardiac blood, and gut were collected from the two dead birds at postmortem examination. Bacteriologic investigations were performed using selective (MacConkey agar, Oxoid, Milan, Italy) and nonselective (blood agar, Oxoid) media in aerobic and anaerobic conditions at 37 C for 24 hr. Fresh smears were prepared from the fecal samples from the
five affected breeders and from the intestinal content of the two dead birds for direct microscopic examination. Each sample was also observed after flotation in saturated NaCl solution with 650 g/L glucose. Histology. Samples of liver were obtained during postmortem examination of the two young finches. For histologic analysis, the blocks of tissue were collected and fixed in 10% neutral buffered formalin. The samples were embedded in paraffin wax, sectioned at 4 mm and stained by hematoxylin and eosin (H&E). Virology. Feather quills were cut and mechanically homogenized in a few drops of distilled water for negative-staining electron microscopy (EM) examination. The method used (15) is based on concentration of viral particles through Airfuge (Beckman Coulter Inc., Brea, CA) ultracentrifugation. Grids were stained with 2% sodium phosphotungstic (NaPT), pH 6.8, for 1.5 min and examined at 19–30000 with a Tecnai G2 Spirit transmission electron microscope (TEM; FEI, Eindhoven, The Netherlands) operating at 85 kV. Viral particles were identified based on their morphologic characteristics. Samples of liver, spleen, gut, and feather from the two finches examined postmortem were screened by PCR to evaluate infections by members of genus Circovirus and avian polyoma virus (APV). Moreover, a PCR assay was used to screen scrapings from beak, feathers, and feces from adults with beak and feather disorders. DNA extraction was performed from each sample by using EuroGold Tissue Mini Kit (EuroClone, Milan, Italy) according to the manufacturer’s instructions. The PCRs were carried out in a mixture containing 1 U of platinum Taq polymerase (Invitrogen, Milan, Italy), 20 mM Tris–HCl, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, and 25 pmol of each primer. Up to 400 ng of total DNA from sample tissues were added to the mixture. Primers DCiVf/DCiVr (28) were used for Circovirus detection.
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Table 1. Comparison of the sequenced rep gene fragment with the corresponding portion of representative members of genus Circovirus. GenBank accession numbers and BFDV hosts are reported.A TN+G distance from % Nucleotide identity EC32/ITA/2011 with EC32/ITA/2011
CaCV AJ301633 FiCV DQ845075 PCV1 U49186 PCV2 AF055392 CoCV AF252610 BFDV1 AF311299 Tha BFDV2 FJ685979 Csu BFDV3 EF457975 Nho BFDV4 AY521238 Per BFDV AY521235 Aro
0.564 0.535 1.056 1.056 0.532 0.102 0.049 0.073 0.048 0.053
63.13 64.53 48.50 50.30 64.53 90.78 95.39 93.19 95.39 94.99
A Tha 5 Trichoglossus haematodus; Csu 5 Cacatua sulphurea; Nho 5 Nymphicus hollandicus; Per 5 Psittacus erithacus; Aro 5 Agapornis roseicollis.
A touchdown PCR protocol was set up due to the high difference between melting temperatures of primer DCiVf and DCiVr. Cycling conditions were as follow: a 5 min denaturation step at 94 C; 14 cycles performed, each with 30 sec of denaturation at 94 C; 30 sec of annealing at 55 C for the first cycle, decreasing by 0.5 C for each of the next cycles; and 1 min of extension at 72 C. A further 21 cycles were conducted as follows: 94 C for 30 sec, 49 C for 30 sec, and 72 C for 1 min, with a final extension at 72 C for 10 min. Primers and cycling conditions previously described (17) were used for APV detection. The PCR products were visualized after agarose gel electrophoresis and 0.5 mg/ml ethidium bromide staining. Cloning and sequence analysis. Two PCR amplicons generated from the liver of a young finch and from the beak of a breeder finch were selected, purified, cloned, and sequenced. The PCR products were purified using the EuroGold Cycle-Pure Kit (EuroClone). Each purified product was cloned in pGEMH-T Easy cloning vector (Promega, Milan, Italy), according to the manufacturer’s protocol. Three to five positive clones were selected for each cloning experiment, and their inserts were sequenced using the Big Dye Terminator v3.1. Sequence determination was performed on an Applied Biosystem ABI 3100 at the facilities of Bmr Genomics (Padua, Italy). The nucleotide sequences from each cloned sample were assembled by using the SeqManII program (DNAStar, Madison, WI). Identification of the product was carried out by comparison with the sequences in GenBank by BLAST. Alignment between the two sequences was performed by ClustalW algorithm (gap open penalty 5 10, gap extension penalty 5 5) implemented in MEGA5 (30). Because the sequences were 100% identical to each other, only one of them was selected and aligned with the corresponding fragment of the rep gene from a representative panel of porcine circovirus (PCV), finch circovirus (FiCV), canary circovirus (CaCV), columbid circovirus (CoCV), and BFDV. The BFDV strains were selected to represent strains identified in each family of the Psittaciformes order. The sequences are listed in Table 1. Alignment was performed by ClustalW with the above-mentioned parameter and with manual editing. The best model for phylogenetic analyses was determined by using Modeltest (24) implemented in FindModel (available online at http://www.hiv.lanl.gov/content/ sequence/findmodel/findmodel.html). The selected model was Tamura– Nei (TN)+G, with gamma distribution shape (a 5 0.49). MEGA was also used to calculate pairwise and group nucleotide and phylogenetic (according to the TN model) distances among sequences. Maximum likelihood phylogenies of the BFDV genomes were inferred using PHYML (10) with 1000 nonparametric bootstrap replicates. The sequence of the analyzed rep gene fragment has been submitted to GenBank with the accession number JX131620.
Fig. 2. Light microscopy images of a liver section. Intranuclear Cowdry type B inclusion bodies (In) and necrosis area (N). H&E 10003 magnification. RESULTS
At postmortem examination, the birds appeared dehydrated and underweight. The liver appeared congested and enlarged. No other gross lesions were evident. Specific bacterial and parasitic pathogens were not detected, as all tests gave negative results. The histologic sections of the livers showed intranuclear Cowdry type B inclusion bodies and chromatin marginalization. Necrotic cells scattered throughout the liver parenchyma were also observed (Fig. 2). Electron microscopy observation of feather quill homogenates revealed the presence of few scattered, small (18–20 nm) icosahedral viral particles without envelope, morphologically related to circovirus (Fig. 3).
Fig. 3. Ultramicrograph of virions observed in the feather quill extract. Icosahedral particles of about 18–20 nm diameter morphologically resembling circovirus. Negative staining 2% NaPT, pH 6.8. FEI Tecnai G2 Spirit TEM at 85 kV. Bar 5 100 nm.
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DISCUSSION
Fig. 4. Maximum likelihood tree of the analyzed rep gene fragment. Internal labels contain the species abbreviation, the GenBank accession number, and, for BFDV, the host abbreviations. Significant bootstrap values are indicated for the main nodes. Tha 5 Trichoglossus haematodus; Csu 5 Cacatua sulphurea; Nho 5 Nymphicus hollandicus; Per 5 Psittacus erithacus; Aro 5 Agapornis roseicollis.
PCR for APV was always negative. On the contrary, PCR for the Circovirus genus generated an amplicon of the expected size (550 bp) from the two liver samples and from all the tested samples from young finches and breeders. Circovirus sequence analysis. Two amplicons were cloned and sequenced. The resultant sequences were identical to each other. Upon BLAST analysis, the best match (97%) was found for the correspondent sequence of the rep gene of a BFDV identified in an African grey parrot (Psittacus erithacus; accession EU810207). Therefore, the virus identified in this study, strain EC32/ITA/ 2011, was characterized as Circovirus. To assess the phylogenetic relationships of the rep gene sequence of EC32/ITA/2011 with other circoviruses, the sequence was aligned with a representative panel of corresponding sequences from BFDV, CaCV, CoCV, FiCV, and PCV retrieved in GenBank. The phylogenetic analysis revealed that EC32/ITA/2011 was closely related to BFDV, whereas it was more distant from the reported FiCV and CaCV sequences, with strong bootstrap support (Fig. 4). Pairwise distances (computed by the TN+G model) between EC32/ ITA/2011 and FiCV and CaCV were 0.535 and 0.564, respectively. By contrast, the mean pairwise distance between EC32/ITA/2011 and the sequences belonging to the BFDV group was 0.065, with nucleotide identity ranging from 91 (Trichoglossus haematodus BFDV) to 95% (P. erithacus BFDV; Table 1).
The histologic findings observed in the liver sections in the young finches, and specifically the finding of inclusion bodies, highlighted a viral infection. Since Polyomavirus, which may cause very similar lesions in the beak and liver of Estrildidae, was not detected, we hypothesize an etiologic role of Circovirus, even if it is known that it also occurs subclinically (32). Nevertheless, other pathogens, such as Macrorhabdus ornithogaster, Candida albicans, Atoxoplasma spp., and Chlamydia psittaci, should be considered in the differential diagnosis, even if they are not commonly associated with the signs and lesions observed in the outbreak. In particular, M. ornithogaster (19) and C. albicans (16) are responsible for mortality in young birds and nestlings, but symptoms, such as regurgitation and vomiting, and lesions in the upper gastrointestinal tract were not observed. Actually, the liver lesions observed at postmortem examination (i.e., enlargement and congestion) resembled those caused by Atoxoplasma spp., but we did not observed in the histologic sections the sporozoites, that are typically found in the liver (25). Chlamydia psittaci is not frequently found in finches, but it has been described recently in canaries (4). It generally causes severe hepatitis and perihepatitis and liver necrosis (1,11). Little is known about the characteristics of Circovirus infections in nonpsittacine birds, and even the associated clinical signs are not yet well defined. A member of the genus Circovirus was identified in a flock of Gouldian finches (24), and it was then considered a novel Circovirus (FiCV) (34). The infection was associated with a respiratory syndrome characterized by lethargy, nasal discharge, severe dyspnea, and death. Previously, the detection of circovirus-like particles was associated with feather loss and liver necrosis in young Zebra finches (21). In other nonpsittacine species, Circovirus was associated with a ‘‘black spot’’ condition characterized by abdominal enlargement and gall bladder congestion in neonatal canaries (8). In adult canaries, the infection was found to be associated with lethargy, anorexia, and feather loss followed by death (35). The virus identified from canaries was later considered a novel Circovirus because of its phylogenetic distance from BFDV and other avian circoviruses (23). Beak pathologies have never been associated with Circovirus infection in Gouldian finches and other nonpsittacine species, whereas they are typical of PBFD in psittacine birds (32). Although more rarely detected than feather disorders, beak abnormalities are observed in the chronic course of PBFD, and they are relatively common in some species, such as cockatoos (7,12,20). The symptoms we observed in the Gouldian finches primarily resemble PBFD rather than the clinical signs described in Circovirus infection in nonpsittacine species. In particular, feather loss was found, but the most evident signs were observed in the beaks of the adult. The lack of evident beak abnormalities in the younger finches was probably due to the different course of the disease. Because the disease progressed more rapidly in the young than in their adult counterparts, the former probably died before developing beak disorders. Moreover, sequence analyses highlighted a substantial identity between the rep gene of EC32/ITA/2011 and those of BFDV, whereas it appeared more distant from FiCV and CaCV. Considering that recent studies (14) evidenced changes and mutations, in particular in important regions of the normally conserved rep gene and not of the cp gene, the possibility of an interspecific spread of BFDV to Gouldian finches, causing the typical clinical features of PBFD, could be suggested. Gouldian finches showed a more rapid course in the development of clinical signs, in particular in the progression of beak lesions with respect to psittacine species. This relatively rapid onset may be due
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to a difference in behavior of the virus in an unusual host such as the Gouldian finch, but the data available at this time are not enough to verify such hypothesis; therefore, further investigations are currently in progress. How the Gouldian finch flock may have acquired the infection remains unclear. The owner has often come in contact with other finch and psittacine bird farmers, and he attended local bird fairs. Therefore, he may have played a role as a vehicle of the virus, considering the widely recognized high stability of BFDV in the environment (31). Nonetheless, all finches were reared outdoors for several months a year, so contact with other potential carriers of the virus cannot be excluded. Interestingly, monk parakeets (Myiopsitta monachus) live as wild birds a few kilometers from the finch aviary; they were probably previously reared as pet birds who, after escape, adapted to wild life and reproduced. Because monk parakeets have a high dispersal potential (9), some of them could have been in proximity of the Gouldian finch flock. However, the circulation of BFDV among the colony has not been verified to date. REFERENCES 1. Andersen, A. A., and D. Vanrompay. Avian chlamydiosis (psittacosis, ornithosis). In: Diseases of poultry, 12th ed. Y. M. Saif, ed. Blackwell Publishing, Ames, IA. pp. 971–986. 2008. 2. Cathedral-Ortiz, L., B. Kurenbach, M. Massaro, K. McInnes, D. H. Brunton, M. E. Hauber, D. P. Martin, and A. Varsani. A new isolate of beak and feather disease virus from endemic wild red-fronted parakeets (Cyanoramphus novaezelandiae) in New Zealand. Arch. Virol. 155:613–620. 2010. 3. Chen, C. L., P. C. Chang, M. S. Lee, J. H. Shein, S. J. Ou, and H. K. Shieh. Nucleotide sequences of goose circovirus isolated in Taiwan. Avian Pathol. 32:165–171. 2003. 4. Circella, E., N. Pugliese, G. Todisco, M. A. Cafiero, O. A. E. Sparagano, and A. Camarda. Chlamydia psittaci infection in canaries heavily infested by Dermanyssus gallinae. Exp. Appl. Acarol. 55:329–338. 2011. 5. Duchatel, J. P., D. Todd, J. A. Smyth, J. C. Bustin, and H. Vindevogel. Observations on detection, excretion and transmission of pigeon circovirus in adult, young and embryonic pigeons. Avian Pathol. 35:30–34. 2006. 6. Eisenberg, S. W. F., A. J. A. M. van Asten, A. M. van Ederen, and G. M. Dorrestein. Detection of circovirus with a polymerase chain reaction in the ostrich (Struthio camelus) on farm in the Netherlands. Vet. Microbiol. 95:27–38. 2003. 7. Gerlach, H. Circoviridae—psittacine beak and feather disease virus. In: Avian medicine: principles and application. B. W. Ritchie, J. Harrison, and L. R. Harrison, eds. Wingers Publishing, Lake Worth, FL. pp. 894–902. 1994. 8. Goldsmith, T. L. Documentation of passerine circoviral infection. In: Proc. Annual Conference of the American Association of Avian Veterinarians, Philadelphia, PA. pp. 349–350. 1995. 9. Gonc¸alves da Silva, A., J. R. Eberhard, T. F. Wright, M. L. Avery, and M. A. Russello. Genetic evidence for high propagule pressure and longdistance dispersal in monk parakeet (Myiopsitta monachus) invasive populations. Mol. Ecol. 19:3336–3350. 2011. 10. Guindon, S., and O. Gascuel. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52:696–704. 2003. 11. Harkinezhad, T., T. Geens, and D. Vanrompay. Chlamydophila psittaci infections in birds: a review with emphasis on zoonotic consequences. Vet. Microbiol. 135:68–77. 2009. 12. Jacobson, E. R., S. Clubb, C. Simpson, M. Walsh, C. D. Lothrop Jr., J. Gaskin, J. Bauer, S. Hines, G. V. Kollias, P. Poulos, and G. Harrison. Feather and beak dystrophy and necrosis in cockatoos: clinicopathologic evaluations. J. Am. Vet. Med. Assoc. 189:999–1005. 1986. 13. Katoh, H., H. Ogawa, K. Ohya, and H. Fukushi. A review of DNA viral infections in psittacine birds. J. Vet. Med. Sci. 72:1099–1106. 2010.
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37. Varsani, A., G. K. de Villiers, G. L. Regnard, R. R. Bragg, K. Kondiah, I. I. Hitzeroth, and E. P. Rybicki. A unique isolate of beak and feather disease virus isolated from budgerigars (Melopsittacus undulatus) in South Africa. Arch. Virol. 155:435–439. 2010.
ACKNOWLEDGMENT We thank Giovanni Circella of the University of California, Davis, CA, for reviewing the English in the manuscript.
