Incidence of Pigeon Circovirus in Eurasian Collared-Dove (Streptopelia decaocto) Detected by Nested PCR

Kubíãek O. , L.Taras: Incidence of Pigeon Circovirus in Eurasian Collared-Dove (Streptopelia decaocto) Detected by Nested PCR. Acta Vet. Brno 2005, 74: 361-368. The paper describes the first demonstration of pigeon circovirus in Eurasian collared-dove (Streptopelia decaocto). Nested PCR was used to examine birds of the Columbiformes family. Pigeon circovirus DNA was identified in the bursa of Fabricius of an accidentally caught dove by nested PCR after the second reaction. Impaired feathers and other clinical signs observed could not, however, be attributed to circoviral infection. The sequence of the amplified PCR product middle of the capsid protein gene was compared against all available circovirus sequences. Both the nucleotide and the derived amino acid sequence have a very close similarity to pigeon circovirus sequences. It may therefore be concluded that the sample was demonstrated to contain pigeon circovirus or another very similar virus. We therefore assume that pigeon circovirus may infect doves, too. Whether it is a special subtype or whether doves may be infected by common pigeon circovirus strains cannot be concluded with certainty from the existing results. PCR, circovirus, pigeon, PiCV, Eurasian collared-dove, turtledove, Streptopelia decaocto Members of the Circoviridae family are the smallest viruses known so far. Their genome comprising around 2,000 nucleotides is formed by single-stranded DNA protected by an icosahedral capside without envelope. As circoviruses mostly induce immune deficiency in birds, the clinical symptoms of infectious diseases are often accompanied by and overlap with secondary infections. Therefore circoviruses escaped attention until recently and their demonstration has been relatively new (Woods and Lat imer 2000). Their incidence is currently being demonstrated in an increasing number of avian species (Todd 2000). At the XIth International Congress of Virology held in Sydney in 1999, the Circoviridae family was divided into two genuses: Gyrovirus with Chicken infectious anaemia virus (CAV) and Circovirus (Pr ingle 1999). Genus Circovirus include two porcine ciroviruses Porcine circovirus 1 (PCV-1) and Porcine circovirus 2 (PCV-2) (Tischer et al. 1974; Meehan et al. 1998), and Beak and feather disease virus (BFDV) (Lat imer e t al. 1991). The newly identified avian circoviruses are pigeon circovirus (PiCV) (Mankertz et al. 2000), goose circovirus (GoCV) (Todd et al. 2001), canary circovirus (CaCV) (Phenix et al. 2001), and Duck circovirus (DuCV) (Hat terman et al. 2003). The primary BFDV hosts include over 50 species of the Psittacidae family. The presence of nucleic acid of this virus has recently been demonstrated even in entirely unrelated species such as flightless birds (Ratitae): the ostrich (Struthio camelus), and songbirds (Passeriformes): mynah (Gracula sp.) (Eisenberg et al. 2003; Rahaus and Wolff 2003). Among Streptopelia, illness caused by circovirus has been described in Streptopelia senegalensis (Pass e t al. 1994). These doves had a feather illness bearing a close ACTA VET. BRNO 2005, 74: 361–368 Address for correspondence: MVDr. Ladislav Taras Avian and Exotic Animal Clinic University of Veterinary and Pharmaceutical Sciences Brno Palackého 1-3, 612 42 Brno, Czech Republic Phone +420 541 562 565 E-mail: TarasL@vfu.cz http://www.vfu.cz/acta-vet/actavet.htm

Members of the Circoviridae family are the smallest viruses known so far.Their genome comprising around 2,000 nucleotides is formed by single-stranded DNA protected by an icosahedral capside without envelope.As circoviruses mostly induce immune deficiency in birds, the clinical symptoms of infectious diseases are often accompanied by and overlap with secondary infections.Therefore circoviruses escaped attention until recently and their demonstration has been relatively new (Woods and Latimer 2000).
The primary BFDV hosts include over 50 species of the Psittacidae family.The presence of nucleic acid of this virus has recently been demonstrated even in entirely unrelated species such as flightless birds (Ratitae): the ostrich (Struthio camelus), and songbirds (Passeriformes): mynah (Gracula sp.) (Eisenberg et al. 2003;Rahaus and Wolff 2003).
Among Streptopelia, illness caused by circovirus has been described in Streptopelia senegalensis (Pass et al. 1994).These doves had a feather illness bearing a close resemblance to the psittacine beak and feather disease (PBFD).Due to the fact that the feather illness in Streptopelia senegalensis resembled PBFD, the authors understandably believed that the birds might have been infected with BFDV.Haemagglutination and haemagglutination inhibitory testing helped to rule out BFDV as the etiological agent in feather disease in Streptopelia senegalensis (Raidal and Ridoch 1997).By electron microscopy, the feather homogenate of these birds was shown to contain circovirus-like particles sized 14-17 nm.The authors therefore concluded that the circovirus is distinct from BFDV; the viral species has not been specified yet.
Pigeon circovirus has been identified in domestic pigeons (Columba livia domestica) only (Todd 2000).A circovirus infection was also confirmed by histology in two young wild wood doves (C.palumbus) (Dorrestein et al. 2001) and by histology and electron microscopy in Senegal doves (Streptopelia senegalensis) (Raidal and Ridoch 1997).The viral species has not been specified by genotyping and it is not known whether it is the species of circovirus infected domestic pigeons, too.
It is mainly young pigeons up to 6 months of age who are susceptible to pigeon circovirus infection.The affected birds show anorexia, lethargy, poor race results, diarrhoea, rapid loss of weight, and mortality (Paré et al. 1999).Although feather illness is not a common symptom of pigeon circovirus infection, a case involving a feather illness has been described, too (Woods et al. 2000).Presence of PiCV infection may be assumed already on the basis of anamnesis, clinical development in the flock, and evidence of specific intracytoplasmic botryoid inclusions in lymphoid tissue.The ultimate proofs include direct detection of PiCV by electron microscopy (Woods et al. 1993), by in situ hybridization (Smyth et al. 2001), and recently most often by PCR (Todd et al. 2001;2002) or nested PCR (Soike et al. 2001;Taras et al. 2003).

Materials and Methods
In October 2004, a 130 g free-living Eurasian collared-dove (Streptopelia decaocto) with a spinal lesion and lesions in the breast muscles caused by mauling was presented at the Clinic of Avian, Reptilian, and Small Mammal Diseases.The dove had been found in Brno municipal area.The dove was killed by T 61 ® (Intervet, Unterschleissheim, Germany) due to a poor prognosis.A pathoanatomical examination using the methodology proposed by Dorrestein (1997) was made during which the macroscopically unchanged bursa of Fabricius was taken out to be subjected to nested PCR.

PCR
DNA isolation was performed using frozen samples of the bursa of Fabricius.An Invisorb Spin Tissue Mini Kit (Invitek Berlin) isolation set was employed.The first PCR reaction was performed on 2 µl of DNA isolate, 7 µl of distilled water, and 1 µl of 25 pmol/µl primers and 10 µl of Top-bio PPP Master Mix (Top-bio, Prague).The proof of PiCV DNA was based on primers from the C 1 ORF (open reading frame) region.The first PCR used Cir Sn 1206 (bases 1206 to 1226, 5'-GCAAAACACTGGTTACAATCC -3') and Cir Asn 1917(bases 1934to 1917, 5'-CAGGAGACGRAGGACACG -3') primers selected based on the comparison of all three available pigeon circovirus sequences in the GenBank and the specificity of the PCR product was verified by comparison with the available sequences in the GenBank using BLAST (Basic Local Alignment Search Tool) software (Altschul et al. 1990).
The reaction solution contained 1 µl of a 25 pmol/µl mixture of both primers, 9 µl of re-distilled sterile water, 10 µl of Top-bio PPP Master Mix, and 1 µl of first PCR amplificates.The second reaction ran for 2 min at 94 °C, then for 30 s at 94 °C, for 20 s at 65 °C, 35 times for 30 s at 72 °C and for 8 min at 72 °C.The reaction was evaluated on 1.5% agar gel stained with ethidiumbromide.The size of the amplification product was compared against DNA using mass marker 2 -Log DNA Ladder (New England Biolabs).The presence of a fragment sized 522 bp was regarded as a positive result.Its specificity was verified by sequencing.

Sequencing
The PCR product nucleotides were sequenced in both directions by capillary electrophoresis with a laser detector at a MegaBACE 1000 genetic analyzer (Amersham Biosciences, Sunnyvale, CA, USA) in Genomac company, Prague.The sequencing used the primers Circo2Sn66 and Circo2Asn566 and marked terminators.

Basic post mortem findings
There was an area of 5 cm in diameter with no feathers on the back of the dove.The feather follicles present were damaged.There were lesions due to bites on the ventral side of the body, whose surroundings were contaminated with secretion.Half of the rectrices were missing.The remige were intact.The loss of feathers on the back and in the tail corresponded with an attack by a predator as for direction.Due to defecation problems caused by the spinal lesion, the area around the cloaca was contaminated with droppings.There were haemorrhages in the subcutis in the site of the lesion.A small lesion caused by a bite situated near the end of the sternum was identified on the left side of breast muscles; there was a lesion next to the shoulder joint on the right-hand side of the body.There were haemorrhages in the body cavity.The internal organs were free of macroscopic change.The spine was broken in the lumbar vertebrae area.The wounded dove was female.

PCR
The first PCR was negative even after DNA isolate concentration.After the second PCR a very strong amplified DNA fragment sized about 520 bp was identified (Plate VII, Fig. 1).PCR product sequence 488 nt (GenBank accession number AY887540) accorded nt 1366 -1853 German isolate PiCV (GenBank accession number NC 002361)

Verification of PCR product specificity
The sequence of the amplified product -nt 1366 -1853 accorded German isolate PiCV (GenBank accession number NC 002361) was compared against available sequences in GenBanks using the Blast software.A high degree of homology with all available PiCV sequences was detected while homology with no another viral nucleic acid was detected.Further analysis revealed a 97% homology with PiCV (Mankertz et al. 2000; accession no.NC 002361), 96% with PiCV 9030, 93% with PiCV 7050 (Todd et al. 2001; accession no.AJ298229 and AJ298230), and 90% with PiCV SM 1 (Taras et al. 2003; accession no.AY461810) (Fig. 2).Amino acid sequence of the derived hypothetic partial capside protein -162 amino acid Position 46 -207 of capsid protein of German isolate PiCV (accession no.NP 059530) KDKITLQQATNDFKFGTGIFTFKLADVLTVGLNAPTLKVPFEDYQIALVKVEMRP LGVDITTWKGFGHTVPMYDARLKTFQGQVDLGDDPLMDFDGARKWDLRKGFK RLIRPRPQLTIADLATANQSAATWFSGRNQWIPLQVSGNSLFPQKVNHYGLAFSY

Phylogenetic analysis
The PCR amplified fragment of circovirus DNA from the Eurasian collared-dove (ECD CV) was compared to partial sequences of ORF C1 of the other circoviruses.The GenBank accession numbers and positions of compared parts are given in the legend to Table 1.This comparison revealed the highest degree of homology with PiCV (Fig. 3).It is evident that

Discussion
The BLAST software has helped us to find a high degree of homology with PiCV only.No conclusive homology with any other circovirus including the most closely related canary circovirus has been identified.The constructed phylogenetic tree shows that the sequence of circoviral DNA isolated from an Eurasian collared-dove differed from the sequences of other pigeon circoviruses less than the variability between the PiCV described as yet.We therefore assume that we have demonstrated PiCV in the Eurasian collared-dove.Based on the high degree of homology with PiCV it may also be hypothesized that the circovirus was not a special subtype, but common PiCV infecting pigeons.Confirmation of this assumption will nevertheless require further data.It seems that we are not talking of another circovirus like those that they have recently been identified in geese (Todd et al. 2001), canaries (Phenix et al. 2001) andducks (Hatterman et al. 2003), but of a finding that like BFDV, a known virus may infect further bird species.
Although feather illness is not a common sign of pigeon PiCV infection, such cases have already been reported in two young wild wood doves (C.palumbus) (Dorrestein et al. 2001), Senegal doves (Streptopelia senegalensis) (Raidal and Ridoch 1997) and in domestic pigeons (Woods et al. 2000).On the other hand, in these cases virus species have not been explicitly characterized by genotyping.It is not clear if these viruses agree with virus reported as PiCV.PiCV have not been demonstrated definitively in any free-living bird yet.One possible explanation is the one by Raidal and Riddoch (1997).According to these authors, infected birds become an easy prey to predators escaping thus examination.The lesions found in the dove we examined are a proof to this as the dove, too, has become a victim to a predator.Unlike previously detected circovirus infection in the Senegal dove  1.The phylogram was generated from aligned, edited sequence data using the Align software (Vector) (Streptopelia senegalensis) (Raidal and Riddoch 1997), the Eurasian collared-dove examined by us was free of symmetric dystrophic change in feathers typical for PBFD.Severity and scope of clinical signs of circoviral infection nevertheless partly depend on the infected species (Dorrestein et al. 2001).The infection in the Eurasian collared-dove may have been in an initial stage and may have thus failed to show in the form of a feather illness.
Provided pigeon circovirus may infect the Eurasian collared-dove, the birds may be a reservoir of the virus in the wild.Free-living doves may therefore be one of the possible sources of infection of domestic pigeons.Table 1.Cap gene sequences used to draft the phylogenetic tree

Fig. 2 .
Fig. 2. Comparison of the sequence of the amplified fragment of circovirus DNA from the Eurasian collared-dove (ECD CV) (middle of capsid protein gene) against available PiCV sequences