Epornitic of avian pox in common buzzards (Buteo buteo): virus isolation and molecular biological characterization Tiziana Rampin, Giuliano Pisoni, Giovanni Manarolla, Daniele Gallazzi, Giuseppe Sironi To cite this version: Tiziana Rampin, Giuliano Pisoni, Giovanni Manarolla, Daniele Gallazzi, Giuseppe Sironi. Epornitic of avian pox in common buzzards (Buteo buteo): virus isolation and molecular biological characteri- zation. Avian Pathology, Taylor & Francis, 2007, 36 (02), pp.161-165. 10.1080/03079450701216647. hal-00540073 HAL Id: hal-00540073 https://hal.archives-ouvertes.fr/hal-00540073 Submitted on 26 Nov 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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Avian Pathology For Peer Review Only Epornitic of avian pox in common buzzards (Buteo buteo): virus isolation and molecular biological characterization Journal: Avian Pathology Manuscript ID: CAVP-2006-0097.R2 Manuscript Type: Original Research Paper Date Submitted by the 06-Dec-2006 Author: Complete List of Authors: Rampin, Tiziana; Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria Pisoni, Giuliano; Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria Manarolla, Giovanni; Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria Gallazzi, Daniele; Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria Sironi, Giuseppe; Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria Avipoxvirus, molecular biological characterization, common Keywords: buzzard, pox epornitic E-mail: [email protected] URL: http://mc.manuscriptcentral.com/cavp Page 1 of 15 Avian Pathology Cavp-2006-0097.R Epinortic of avian pox in common buzzards ( Buteo buteo ): virus isolation and molecular biological characterization Tiziana Rampin*, Giuliano Pisoni, Giovanni Manarolia, Daniele Gallazzi, and Giuseppe Sironi. For Peer Review Only Dipartimento di Patologia Animale, Igiene e Sanita Pubblica Veterinaria, via Celoria 10 Milano 20133, Italy. Running title: avian pox in common buzzards Figure 1 to be in black and white in the printed version (see black and white file), with colour only online • To whom correspondence should be addressed. Tel.+39 02 50318104. Fax. +39 02 50318106 Received: 10 July 2006 1 E-mail: [email protected] URL: http://mc.manuscriptcentral.com/cavp Avian Pathology Page 2 of 15 Cavp-2006-0097.R1 Tiziana Rampin, Giuliano Pisoni, Giovanni Manarolia, Daniele Gallazzi, and Giuseppe Sironi Epinortic of avian pox in common buzzards ( Buteo buteo ): virus isolation and molecular biological characterization Abstract For Peer Review Only Six common buzzards from a bird rescue centre showed wart-like lesions on their toes. The lesions consisted of multiple crusty and proliferative nodules surrounded by skin swelling. Histologically, epithelial cell hyperthrophy and hyperplasia with ballooning degeneration and large intracytoplasmic inclusion bodies consistent with avipox virus infection were seen. The virus was isolated in embryonated chicken eggs. Positive CAMs and samples of skin lesions were submitted for PCR. The sequencing of the amplicons obtained with primers for 4b core protein revealed a 100% identity of the isolated poxvirus with pigeonpox virus TP2. 2 E-mail: [email protected] URL: http://mc.manuscriptcentral.com/cavp Page 3 of 15 Avian Pathology Introduction Pox is a worldwide disease caused by avian pox viruses (APV) belonging to the genus Avipoxvirus of the family Poxviridae . It is reported in numerous domestic and wild avian species (Bolte et al. , 1999). Gross lesions in both the cutaneous and diphteric/pharingeal forms are usually sufficient to suspect a pox infection (Tripathy and Reed, 2003), and the histological featuresFor (Pass, Peer 1996) are regarded Review as definitely confirmat Onlyory for the diagnosis in any affected species. Furthermore, the number of the species and tentative species listed in the genus Avipoxvirus (www.virustaxonomyonline.com) is much smaller than the number of bird species naturally infected by pox and consequently the attribution of an APV strain to a novel species within the genus Avipoxvirus requires biologic, antigenic and genetic characterization (Kim et al. , 2003). Although among APVs antigenic and immunological differences as well as differences in host specificity have been reported for domestic fowl (Tripathy and Reed, 2003), little information is available about pox strains in wild birds. As for birds of prey, pox has been reported in several species of the families Falconidae and Accipitridae (Bolte et al. , 1999). Nevertheless, the reports of pox in common buzzard ( Buteo buteo ) are relatively rare (Loupal et al. , 1985; İzmen and Dorrestein, 2002). The present report describes an epornitic of avian pox in captive common buzzards with molecular characterization of the APV isolates. Six common buzzards were housed in an outdoor flight cage of a rehabilitation centre for wild birds in Northern Italy. They came from different areas within Italy and were admitted to the rescue centre in different periods between January and July 2005. One buzzard died in September 2005 and was submitted to our laboratory for necropsy. The referring veterinarian notified the presence of wart-like lesions on the toes of all the buzzards of that cage. This buzzard showed diffuse mycotic air sacculitis. Furthermore, proliferative skin lesions were observed on both feet. The suspicion of a pox infection was promptly forwarded 3 E-mail: [email protected] URL: http://mc.manuscriptcentral.com/cavp Avian Pathology Page 4 of 15 to the rehabilitation centre staff who decided to humanly euthanize the remaining five buzzards of that aviary when the suspicion was confirmed. These five birds also were presented for necropsy. Materials and Methods For Peer Review Only Sample collection. At necropsy, samples of skin nodules were collected from all six buzzards for histopathology, and were frozen at –80°C to perform virus isolation and DNA extraction. Notwithstanding the lack of other gross lesions, samples of liver, spleen, lung and heart were collected for histopathology. Virus isolation. Tissue samples from each bird were minced and ground with sterile quartz sand using a mortar and pestle, suspended in a balanced salt solution containing 50 IU/ml penicillin and 50 µg/ml streptomycin. Following low speed centrifugation at 100 x g for 10 min, 0.1 ml of the supernatant of the six suspensions was inoculated on the chorioallantoic membranes (CAM) of 11-day-old chicken embryos from a commercial line. The inoculated eggs were incubated at 37°C for 7 days and then examined for pocks on the CAM. No further passages were carried out. Histopathology . Tissue samples collected from all six buzzards and the CAMs were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 4 µm and stained with haematoxylin and eosin. Feulgen stain for detection of DNA was performed on skin and CAM sections. 4 E-mail: [email protected] URL: http://mc.manuscriptcentral.com/cavp Page 5 of 15 Avian Pathology DNA extraction and PCR amplification . DNA was extracted from 25 mg of the CAM with evident pocks and from the skin lesions of all the six buzzards by QIAamp DNA Mini Kit (Qiagen, Italy, Milan) following the manufacturer’s guidelines. Tissue digestion with proteinase K was performed at 56°C for 2 h for the CAM and at 56°C overnight for the cutaneous lesions. The DNA concentration was measured fluorometrically and DNA samples were stored at -80°CFor until Peeranalysis. Review Only APV specific PCR was performed using a primer pair chosen according to the published 4b core protein gene sequence of fowlpox virus strain HP444 (Lee and Lee, 1997) and as described by Lüschow et al. (2004). Five µl of the amplified PCR products were separated on 2% agarose gel electrophoresis and stained with ethidium bromide. PCR products with the specific size (578bp) were sequenced by CRIBI Services (CRIBI, Padova, Italy) on an ABI377 sequencer by using the ABI PRISM dye-terminator cycle sequencing ready reaction kit with Amplitaq DNA polymerase (Perkin-Elmer, Applied Biosystems). The sequence was submitted to the GenBank database (accession number EF016108). After manual editing and excluding primer regions, the 4b gene sequences (523 bp) were aligned (ClustalW; Thompson et al. , 1994) with APV sequences available from GenBank and possible genetic relationship and phylogenetic grouping of the different APVs were investigated using the method of neighbour joining with Jukes and Cantor model (Mega version 2.1; Kumar et al. , 2001). The reliability of the tree topologies was tested by bootstraping. Results Gross and microscopic examination of the six buzzards. All six birds were in good body condition and nodular skin lesions were observed on the dorsal, lateral and ventral phalangeal 5 E-mail: [email protected] URL: http://mc.manuscriptcentral.com/cavp Avian Pathology Page 6 of 15 surfaces of both feet. Each foot showed multiple lesions varying from papules to nodules, ranging from