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
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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
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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
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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.
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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
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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 g/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 m and stained with haematoxylin and eosin. Feulgen stain for detection of DNA was performed on skin and
CAM sections.
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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
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surfaces of both feet. Each foot showed multiple lesions varying from papules to nodules, ranging from 0.2 to 1 cm. The larger ones appeared dark brown with crusty or ulcerated surface. Cutaneous lesions were confined to the feet (Figure 1) and no diphtheric lesions were detected on the mucosal surfaces. Severe mycotic air sacculitis was observed in the spontaneously dead buzzard whereas no other gross lesions were detected in the euthanized ones. For Peer Review Only The histological evaluation of the cutaneous lesions revealed marked hyperkeratosis and achantosis associated with multifocal ulcers, cellular debris and large aggregates of cocci.
Most keratinocytes were characterized by ballooning degeneration with large intracytoplasmic
15-20 m, pale eosinophilic inclusions surrounded by a clear halo (Bollinger bodies). The inclusions were positive with Feulgen stain. Dermis was moderately expanded by oedema and infiltrated by high numbers of heterophils and rare lymphocytes and plasma cells. Sections of liver, spleen, lung and heart from euthanized buzzards were unremarkable.
Virus isolation. Macroscopically, signs of viral growth characterized by multifocal, white, opaque pocks on the CAM were obtained from one buzzard and only in one egg.
Nevertheless, histologically evidence of viral replication was focally observed in the CAMs of all the six inocula. The prominent feature was epithelial hypertrophy and hyperplasia with severe ballooning degeneration and relatively rare cytoplasmic inclusions positive for DNA with Feulgen stain. These changes were obviously severest in the unique CAM with pocks.
This CAM was collected and stored at –80°C.
PCR and sequencing. APV-specific DNA was detected in the skin lesion samples from all the six birds and in the infected CAM. The size of all the amplified products (approximately
580 bp) was in agreement with the size of the published FPV 4b nucleotide sequence.
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Consequently, sequencing of the amplified region was performed and the sequences were
compared with the APV 4b nucleotide sequences from GenBank. Sequences analysis revealed
a 100% nucleotide identity of all the amplicons with pigeonpox virus (PGPV), strain TP2.
The derived phylogenetic tree (Figure 2) showed that the sequences in this study formed a
cluster with the aforementioned PGPV-TP2 and with a poxvirus isolated from ostrich (GB 724/01-20) withFor which there Peer was a nucleotide Review identity of 99-100%. Only
Discussion
Pox lesions have been recorded in many diurnal raptors including buzzards. In these birds the
cutaneous lesions are much more common than the diphteric ones (Graham and Halliwell,
1978; Wheeldon et al, 1985; Schoemaker et al, 1998; Mete et al, 2001; İzmen and
Dorrestein, 2002). The presentation of this epornitic was similar to that reported in other
raptorial species with typical pox lesions confined to the feet. Traumatic skin lesions on the
feet are common in captive raptors and sharing contaminated perches can make easier the
lateral spread of the infection (Graham and Halliwell, 1978).
The genetic characterization of this isolate revealed an identity of 100% with the
PGPV-TP2 as demonstrated by sequencing of the PCR products. A poxvirus strain with 4b
gene sequence almost identical to the PGPV-TP2 was isolated from a ostrich (Lüschow et al. ,
2004). Recently, a philogenetical analysis of the P4b locus clustered both of these viruses in
the subclade A2 with turkeypox virus (Jarmin et al. , 2006). Therefore, this strain responsible
for pox epornitic in common buzzards is to be included in the same subclade.
As for the origin of this pox infection, unfortunately we lack information for
completing the infectious history of this epornitic. Nevertheless, the following aspects need to
be considered. The epornitic involved six birds coming from different areas and admitted to
the centre in different months. The cutaneous lesions became manifest in late summer, the
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season with the highest incidence of avian pox in Italy. The staff of the rescue centre stated that the buzzards had not had any contact with turkeys, pigeons and ostriches, so far these being the only reported hosts of APVs belonging to subclade A2 (Jarmin et al. , 2006). It is possible that pox infection may have been introduced into the centre through biting arthropods whose role in the transmission of avian pox is well known (Damassa, 1966; Akey et al ., 1981). Alternatively,For Peer a buzzard may Review have had an inapparent Only infection when it was admitted. In both cases, the infection spread successfully among birds kept together as demontrated by sequence identity of the 6 isolates. However, the centre staff did not report pox lesions in raptors living in other outdoor cages of the facilities.
As for virus isolation, the strain displayed limited growth with evident pocks only on one CAM. Usually, APVs from domestic fowls, canaries and pigeons can be easily isolated in chicken eggs whereas some strains from wild birds are reported to fail propagation in chicken embryos (Tripathy and Reed, 1998; Krone et al. , 2004). However, we cannot exclude that the poor propagation of this strain may have been due to chicken eggs from commercial breeders vaccinated against fowlpox.
In conclusion, genetic analysis increasingly applied to APV strains has been revealing unexpected similarities among APVs isolated from different avian species. The present strain was isolated from a raptorial species where pox is a rarely characterized disease, and it clustered phylogenetically with better known members of APVs such as pigeonpox virus and turkeypox virus (Jarmin et al. , 2006). Therefore, the acquisition of sequence data appears necessary to complete the information about pox epornitics in domestic and wild birds.
Acknowledgements
This work was supported by grants from Regione Lombardia (Italy).
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References
Akey, B.L., Nayar, J.K. & Forrester, D.J. (1981). Avian pox in Florida wildturkeys: Culex
nigripalpus and Wyeomyia vanduzeei as experimental vectors. Journal of Wildlife Diseases, 17For, 597-599. Peer Review Only Bolte, A.L., Meurer, J. & Kaleta, E.F. (1999). Avian host spectrum of avipoxviruses. Avian
Pathology, 28 , 415-432.
Damassa, A.J. (1966). The role of Culex tarsalis in the transmission of fowl-pox virus. Avian
Diseases, 10 , 57-66.
Graham, D.L. & Halliwell, W.H. (1978). Virus diseases of birds of prey. In M.E Fowler.
(1978). Zoo and Wild Animal Medicine (pp.260-261). Philadelphia: W.B. Saunders
Company.
Jarmin, S., Manvell, R., Gough, R.E., Laidlaw, S.M. & Skinner, M.A. (2006). Avipoxvirus
phylogenetics: identification of a PCR length polymorphism that discriminates between
the two major clades. Journal of General Virology, 87 , 2191-2201.
Kim, T.J., Schnitzlein, W.M., McAloose, D., Pessier, A.P. & Tripathy, D.N. (2003).
Characterization of an avianpox virus isolated from an Andean condor (Vultur gryphus) .
Veterinary Microbiology, 96 , 237-246
Krone, O., Essbauer, S., Wibbelt, G., Isa, G., Rudolph, M. & Gough, R.E. (2004).
Avipoxvirus infection in peregrine falcons ( Falco peregrinus ) from a reintroduction
programme in Germany. The Veterinary Record, 154 , 110-113.
Kumar, S., Tamura, K., Jakobsen, I.B. & Nei, M. (2001). MEGA2: molecular evolutionary
genetics analysis software. Bioinformatics, 17 , 1244-1255.
9 E-mail: [email protected] URL: http://mc.manuscriptcentral.com/cavp Avian Pathology Page 10 of 15
Lee, L.H., & Lee, K.H. (1997). Application of the polymerase chain reaction for the diagnosis
of fowl poxvirus infection. Journal of Virological Methods, 63 , 113-119.
Loupal, G., Schönbauer, M. & Jahn, J. (1985). Pocken bei Zoo- und Wildvögeln. Zentralblatt
für Veterinärmedizin. Reihe B, 32 , 326-336.
Lüschow, D., Hoffmann, T. & Hafez, H.M. (2004). Differentiation of Avian Poxvirus Strains on the BasisFor of Nucleotide Peer Sequences Review of 4b Gene Fragment. Only Avian Diseases, 48 , 453-462. Mete, A., Borst, G.H.A. & Dorrestein, G.M (2001). Atypical poxvirus lesions in two
Galapagos doves (Nesopelia g. galapagoensis). Avian Pathology, 30 , 159-162.
İzmen, İ. & Dorrestein, G.M. (2002) Avipox in a Common Buzzard (Buteo buteo) . Turkish
Journal of Veterinary and Animal Sciences, 28 , 1193-1195.
Pass, D.A. (1996). Integumentary System. In C. Riddell (1996). Avian Histopathology 2nd
edn (pp 219-230). Saskatchewan, Canada: American Association of Avian Pathologists.
Schoemaker, N.J., Dorrestein, J.M. & Lumeij, J.T. (1998). An avipoxvirus infection in a
goshawk (Accipiter gentilis) . Avian Pathology, 27 , 103-106.
Thompson, J.D., Higgins, D.G. & Gibson, T.J. (1994). CLUSTAL W: improving the
sensitivity of progressive multiple sequence alignment through sequence weighting,
position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22 ,
4673-4680.
Tripathy, D.N. & Reed, W.M. (1998). Pox. In D.E. Swayne, J.R. Glisson, M.W. Jackwood,
J.E. Pearson & W.M. Reed (1998). Isolation and Identification of Avian Pathogens 4 th edn
(pp.137-140). New Bolton Centre, PA: American Association of Avian Pathologists.
Tripathy, D.N. & Reed, W.M. (2003). Pox. In Y.M. Saif, H.J. Barnes, J.R. Glisson, A.M.
Fadly, L.R. McDougald, & D.E. Swayne. (2003). Diseases of Poultry 11th edn (pp.253-
269). Ames: Iowa State Press.
10 E-mail: [email protected] URL: http://mc.manuscriptcentral.com/cavp Page 11 of 15 Avian Pathology
Wheeldon, E.B., Sedgwick, C.J. & Schulz, T.A. (1985). Epornitic of avian pox in a raptor
rehabilitation centre. Journal of the American Veterinary Medical Association, 187 , 1202-
1204.
For Peer Review Only Figure legends
Figure 1 . Nodular to wart-like lesions on the foot of a common buzzard (Buteo buteo).
Figure 2. Phylogenetic analysis of nucleotide sequences of the 4b core protein gene of APV
isolated in this study (named as PA 153/05) and of published APVs sequences. The tree was
obtained by neighbour-joining method calculated with the Jukes and Cantor model. Bootstrap
testing of phylogeny was performed with 1000 replications and values equal or greater than
60 are indicated on the branches (as percentage). The length of each bar indicates the
amount of evolution along the horizontal branches as measured by substitution per site.
Reference sequences are available from GenBank under accession numbers: FPV HP444
(M25781), FPV HP-B (AY530302), GB134/01 (AY530304), DD1258 (AY530307), GB320/02
(AY530308), PPV TP-2 (AY530303), WCV33-03 (DQ131892), WCV1419-03 (DQ131901),
GB724/01-20 (AY530305), GB362/02 (AY530306), GB724/01-22 (AY530309), GB182/98
(AY530310), APIII (AY530311).
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For Peer Review Only
Figure 1. Nodular to wart-like lesions on the foot of a common buzzard ( Buteo buteo ). 80x106mm (600 x 600 DPI)
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For Peer Review Only
Figure 1. Nodular to wart-like lesions on the foot of a common buzzard ( Buteo buteo ). 80x106mm (300 x 300 DPI)
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FWPVD FWPVM FWPVN FWPV174 100 FWPVHPB A1 FWPVFP9 TKPV13401 FWPVVR250 SRPVDD1258 73 PGPVTP2 PGPVP For Peer Review100 OSPV OnlyA2 TKPV66 100 A PA153/05 buzzard 99 TKPV98 ABPV A3 100 FLPV1381 FLPV36202 A4 GTPVA311 GTPVA310 SCPV18298 98 GTPV256 CNPVATCC CNPVV B1 SRPV9037 SRPV32002 79 HOPV1252 87 CNPV72401 100 SRPV23 B CNPV1445 99 SLPV
100 PGPVB7 B2 100 PGPV950 PRPV C C MCPV 100 AGPV MOCV
0.05
Figure 2. Phylogenetic tree of nucleotide sequences of the 4b core protein gene of APV isolated in this study (named as PA 153/05), reference APVs sequences and MOCV orthologue sequence, rooted on MOCV. The tree was obtained by neighbour-joining method calculated with the Jukes and Cantor model. Bootstrap testing of phylogeny was performed with 1000 replications and values equal or greater than 70 are indicated on the branches (as percentage). The length of each bar indicates the amount of evolution along the horizontal branches as measured by substitution per site. Avipoxvirus clades A-C and subclades are labelled. Reference sequences are available from Jarmin et al. (2006).
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CNPVATCC SRPV9037 100 HOPV1165 B1 SRPV23 CNPV1445 FWPV174 FWPVHP1
100 FWPVD For Peer Review OnlyA1 FWPVUS FWPVN FWPVFP9
99 ABPV 99 A3 FLPV1381
95 PGPVP TKPV98 A2 90 100 TKPV66 PA153/05 buzzard MOCV
0.1
Figure 3. Phylogenetic tree of fpv139 nucleotide sequences of APV isolated in this study (named as PA 153/05), reference APVs and MOCV orthologue sequence, rooted on MOCV. The tree was obtained by neighbour-joining method calculated with the Jukes and Cantor model. Bootstrap testing of phylogeny was performed with 1000 replications and values equal or greater than 70 are indicated on the branches (as percentage). The length of each bar indicates the amount of evolution along the horizontal branches as measured by substitution per site. Avipoxvirus clades A-B and subclades are labelled. Reference sequences are available from Jarmin et al. (2006).
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