Avian Pathology ( 2001) 30, 605–611
A diagnostic study on columbid circovirus infection Dirk Soike1*, Kim Hattermann2, Kerstin Albrecht1, Joaquim Segal´es3, Mariano Domingo3, Cornelia Schmitt2 & Annette Mankertz2 1
SVLA Potsdam, Pappelallee 20, 14469 Potsdam, Germany, 2P24 ( Xenotransplantation), Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany, and 3Histologia i Anatomia Patol`ogia, Facultat de Veterin a` ria, Universitat Aut`onoma de Barcelona, 08193 Bellaterra ( Barcelona), Spain
A systematic study was performed to examine the frequency of columbid circovirus ( CoCV) infection in diseased young pigeons submitted for necropsy and its relevance to pigeon health. Existing diagnostic methods were compared. Among 176 diseased young pigeons examined, CoCV infection was detected in 83 cases using negative contrast electron microscopy. Histopathological examination allowed a clear diagnosis in only 42 pigeons. Therefore, a polymerase chain reaction assay and an in situ hybridization test were developed as additional diagnostic tools. CoCV is by far the most frequently detected infectious agent in diseased young pigeons. Infected pigeons reveal a broad range of concurrent infections. Pathological findings suggest an immunosuppressive effect of CoCV.
Introduction Circoviruses are the smallest animal viruses known. They are non-enveloped, isometric particles ( diameter, 12 to 26 nm) and contain a single-stranded DNA genome. The family Circoviridae includes chicken anaemia virus ( CAV) as the only member of the genus Gyrovirus, and porcine circovirus ( PCV) as well as beak and feather disease virus ( BFDV) as members of the genus Circovirus ( McNulty et al., 2000; Todd, 2000) . This classification will probably be updated, because PCV type 2 ( PCV2) ( Hamel et al., 1998; Meehan et al., 1998; Morozov et al., 1998; Mankertz et al., 2000a) and columbid circovirus ( CoCV) ( Mankertz et al., 2000b) have been isolated and sequenced recently and proposed as new members of the genus Circovirus. A virus with 67% nucleotide identity with the replication associated protein gene of CoCV has been demonstrated in canaries ( Todd et al., 2001). CoCV shows homology to BFDV, PCV1 and PCV2 in genome structure, as well as in sequence and structural elements of the putative origin of replication in the amino acid sequence of the replicase and in the putative capsid protein. A common characteristic of PCV2 infection in pigs, BFDV infection in psittacine birds and CAV infection in chickens is damage to lymphoreticular
tissue. A recently described circovirus infection in geese, related to a runting syndrome, is associated with a very similar pathological pattern ( Soike et al., 1999). Immunosuppression is therefore thought to be a feature of circovirus infections in different species. Nayar et al. ( 1999) demonstrated the presence of a circovirus PCR product in lung tissue from cattle with respiratory disease or lung tissue of aborted bovine foetuses. Circovirus-like particles have been observed in our laboratory in the lymphoreticular tissue of muscovy ducks and pekin ducks, and in a faecal sample from a cow with diarrhoea. Little is known about the prevalence of circoviruses in wild bird populations. Recently, a circovirus infection in black-backed gull ( Larus dominicanus) has been described ( Twentyman et al., 1999), suggesting that circoviruses may be more widespread in avian species than previously recognized. The first cases of circovirus infection in pigeons were reported from the US ( Schmidt, 1992; Woods et al., 1993). Since then, the agent has been identified in diseased pigeons from South Africa ( Gerdes, 1993), Australia, Canada ( Woods et al., 1994), Great Britain ( Smyth & Caroll, 1995) , Germany ( Soike, 1997) and Belgium ( Duchatel et al., 1998). There are detailed descriptions of the pathology of circovirus infection in pigeons from
* To whom correspondence should be addressed. E-mail:
[email protected] Received 24 October 2000. Accepted 15 March 2001. ISSN 0307-9457 ( print)/ISSN 1465-3338 ( online)/01/060605-07 © 2001 Taylor & Francis Ltd DOI: 10.1080/03079450120092099
606 D. Soike et al.
individual cases ( Woods et al., 1994; Pare´ et al., 1999). However, little is known about the prevalence of the infection and its relevance to pigeon health. Smyth ( 1995) found histopathological evidence for circovirus infection in 14 of 24 young racing pigeons examined. Circovirus infection was detectable in 10 of 17 lofts, indicating a wide distribution of the infection among young racing pigeons in Northern Ireland. This study describes a systematic study on circovirus infection in diseased young pigeons from a German region, and the development of diagnostic techniques that can be used to demonstrate presence of CoCV in infected pigeons. Materials and Methods To obtain data on the prevalence of CoCV infection in diseased young pigeons, 176 birds aged between 3 weeks and about 9 months were examined using negative-staining electron microscopy ( EM) and histopathology. Animals were submitted for necropsy with a history of increased rearing losses and/or various clinical signs in individual birds. A full necropsy and diagnostic evaluation, including examinations for pathogenic bacteria, viruses, parasites, fungi and chlamydia, were performed in each case. Selected organs of each animal ( lung, trachea, liver, kidney, intestine, bursa, thymus and spleen) were examined using negative-staining EM and histopathology following standard procedures. Electron microscopy Tissue homogenates were diluted 1:5 in phosphate-buffered saline ( PBS), sonicated for 40 sec at 20 kHz and clarified for 15 min at 3000 ´ g. The supernatant was used for direct preparation and particle enrichment. A 400 mesh Zapon-covered and carbon-coated copper grid was placed on a drop of the supernatant or directly on the tracheal mucosa for 7 min. Following removal of excess liquid and a washing step in distilled water, the specimen was stained with 2% phosphotungstic acid in PBS ( pH 6.0) for 7 min and air dried. For particle enrichment, the supernatant was centrifuged for 12 min at 120 000 ´ g directly on the microscopic grid using an air-driven ultracentrifuge ( Beckman Airfuge) with an A-110 Rotor. The enriched specimen was then stained as already described. Specimen grids were examined with a Jeol JEM 1010 electron microscope at 40 000-fold magnification and 80 kV. Polymerase chain reaction The newly established nested polymerase chain reaction ( PCR) was used for examination of 41 selected tissue samples for the presence of CoCV DNA ( Table 1). These samples included 21 lymphoreticular tissue samples from 16 diseased young pigeons, 12 additional tissue samples from one of the young pigeons, as well as seven samples from two pigeon embryos and one sample from a freshly embryonated egg submitted for diagnostic evaluation with diseased young pigeons. All samples were also examined using negative-staining EM to compare the reliability of both methods. DNA was prepared from tissue samples as described previously ( Mankertz et al., 2000b). These samples were tested first using primers amplifying a fragment from the highly conserved cytochrome b gene as a control PCR. Only samples proven to be free of inhibitory components were subjected to the CoCV PCR. Since the highest diversity between circoviral genomes from different species is found within open reading frame C1, the putative cap gene, primers were selected from this region of the CoCV genome. A 150 ng sample of DNA isolated from pigeon tissue was included in a reaction containing 0.75 U Taq polymerase, 1 ´ PCR buffer ( Perkin Elmer), 2 mM MgCl2, 200 mM dNTPs, 1 mM each primer and 5% dimethlysulfoxide in a volume of 25 ml. Primer F330 ( bases 1282 to 1299, 59-TGAGGGTGGTCCAAGCAA ) and primer B331 ( bases 1893 to 1874, 59-ACAGGAGGAGTAGCCGTATT) were used in the first-
Table 1. Examination of tissue samples using PCR and electron microscopy Sample number
Pigeon number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
3422/99 3422/99 2935/99 3851/99 3935/99 3941/99 4667/99 4668/99 4667/99 4699/99 4700/99 577/00 1272/00 1274/00
1592/00 1593/00
1594/00 1595/00 1600/00 7/00
Tissue Bursa Thymus Spleen Bursa Bursa Bursa Bursa Spleen Bursa Bursa Bursa Bursa Bursa Thymus Spleen Liver Spleen Kidney Lung Bursa Thymus Trachea Pancreas Intestine Brain Skeletal muscle Ovary Skin Gizzard Heart Thymus/embryo Bursa/ embryo Allantoic sac Spleen/embryo Thymus/embryo Bursa/embryo Allantoic sac Bursa Bursa Bursa Embryonated egg
PCR result
EM result
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + +
– +
– + + +
– + + +
–
– – – – +
– – + +
– – +
– – – – – – – – – – – – – – – – –
round PCR. A 1 ml sample of the amplified DNA was diluted 1:50 and reamplified using primer pair F332 ( bases 1375 to 1392, 59-ATGCGAGCCCATAGTGAT) and B333 ( bases 1711 to 1690, 59-TTAGTGAAGGTGGAAATGAGAC) using the same conditions. Both reactions were incubated at 95°C for 12 min then at 95°C for 20 sec, 53°C for 20 sec and 72°C for 45 sec for 42 cycles, then at 72°C for 10 min. Amplification of the 336 bp second-round product was analyzed by subsequent gel electrophoresis. To ensure that CoCV-specific sequences had been generated, resultant PCR fragments were sequenced directly as described previously ( Mankertz et al., 2000b). Sensitivity of the nested PCR approach was tested with plasmid pTIK, carrying the complete CoCV genome cloned into vector pCR2.1. Serial dilutions of pTIK from 100 fg to 1 ag in 150 ng DNA isolated from circovirus-free PS cells ( porcine kidney cells derived from PK-15 cells) were amplified as already described. Product analysis revealed that 1.3 copies of CoCV were detected in 150 ng template DNA ( data not shown). Comparison of the nucleotide sequence of the cap genes of
Pigeon circovirus infections 607 PCV1 and PCV2, BFDV and CoCV ( using the CLUSTAL W module of McVector) suggested that the primers for the CoCV-specific PCR should not amplify genomic DNA from related circoviruses. To ensure that the nested PCR amplifies CoCV specifically, 150 ng DNA isolated from PCV1-infected cells, spleen from a PCV2-infected pig or liver from a BFDV-infected parrot were tested by PCR with CoCV-specific primers #330/#331 and #332/#333. This did not result in a PCR product ( data not shown), while PCR products were seen after testing of these samples with primers specific for each of the respective infectious agents. This indicated that CoCV was detected using with this nested PCR approach, but the DNA of other circoviruses was not. In situ hybridization To compare the reliability of the newly developed in situ hybridization test ( ISH) and negative-staining EM, 50 selected lymphoreticular tissue samples from 20 diseased young pigeons ( Table 2) were examined using both methods. Tissue sections were placed on Probe On Plus glass microscope slides ( Fisher Scientific, Pittsburgh, PA, USA). A work-station was used to handle the slides, to control the temperature of the hybridization reactions and various incubations, and to minimize reagent consumption. Tissue sections were dewaxed in xylene and rehydrated in graded alcohols to Automotion Buffer ( Biømeda Corp., CA, USA). They were then digested with 0.3% pepsin and incubated with 100% formamide for 5 min at 105°C. Sections were subsequently hybridized to a CoCV-specific, single-stranded, 42-nucleotide probe ( 59-ATAGTTGATGGGGTACTTCTTCATACAATCCGGAGCATCTTC-39, bases 991 to 950) complementary to the 39 end of the rep gene. This DNA probe was end-labelled with digoxigenin. Hybridization was performed for 5 min at 105°C and for 45 min at 37°C. High-stringency washes were performed with saline sodium citrate buffer to ensure a complete match between the target nucleic acid and DNA probe. After the washes, an anti-digoxigenin antibody conjugated to alkaline phosphatase was applied to the sections. Colour was developed with nitroblue tetrazolium dye. Dye reduction to insoluble blue–black formazan indicated areas of probe hybridization. Tissue sections were counterstained with Fast Green, dehydrated, covered with coverslips, and examined. Sections of bursa of Fabricius from a previous case of CoCV infection diagnosed in Germany were used as a positive control for the ISH technique ( Soike, 1997). Negative controls consisted of spleen and bursa sections of a healthy pigeon. To test the specificity of the probe, tissue sections from a pig naturally infected with PCV2 and from a parrot naturally infected with BFDV were tested.
Results Using negative-staining EM, circovirus infection was detectable in 48% ( 83 of 176) of the diseased young pigeons submitted for necropsy. The infected cases were distributed throughout the western part of the German federal state Brandenburg. Because of the seasonal breeding behaviour of the pigeon, 78% of the young pigeons were submitted for necropsy between June and November in the years 1996 to 2000. About 90% of the circoviruspositive young pigeons were detected during these months. Case histories often indicated increased rearing losses. With the age-related involution of the bursa and thymus, the frequency of circovirus detection could be expected to decrease. All circovirus-positive pigeons examined were aged between 2 weeks and approximately 9 months. Therefore, data of all pigeons older than 9 months of age were excluded from further analyses. Specific clinical features that might be attributed to circovirus infection were not reported. On gross examination, circovirus-specific pathological changes were not seen.
Table 2. Examination of tissue samples using ISH and electron microscopy Sample number
Pigeon number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
1288/96 1290/96 1385/96 1454/96 1456/96 1581/96 1685/96 1715/96 1742/96 1759/96 1760/96 1761/96 1766/96 1778/96 1716/96 1951/96 704/97 705/97 875/97 1421/97
Tissue Bursa Thymus Spleen Bursa Thymus Spleen Bursa Spleen Spleen Spleen Bursa Spleen Bursa Thymus Spleen Bursa Spleen Spleen Bursa Thymus Spleen Bursa Thymus Spleen Bursa Thymus Spleen Bursa Thymus Spleen Bursa Thymus Spleen Bursa Thymus Spleen Bursa Thymus Spleen Bursa Thymus Spleen Bursa Thymus Spleen Bursa Thymus Spleen Bursa Spleen
ISH result
EM result
+ + +
+ + + +
– – –
– –
+
+
–
– –
+ + + +
+ +
–
– – –
+ +
+ +
+ +
–
–
+ + + + + + + + + +
+ + + + + + + + + +
– – – – –
– –
+
+ + +
– + + + + + + + + + + + + + + +
–
+
– –
– – + + + + + + + + + + + +
Histopathology Histopathological changes indicative of circovirus infection were restricted to the lymphoreticular tissue. Lesions ranged from follicular hyperplasia and mild lymphocytic necrosis to lymphocytic depletion and generalized histiocytosis. Usually,
608 D. Soike et al.
Figure 1. Bursa of Fabricius of a CoCV-infected young pigeon with numerous inclusion bodies in medullary and cortical bursal follicular cells. Haematoxylin and esoin stain, bar represents 100 mm.
these changes were rather moderate in the spleen and thymus but particularly evident in the bursa of Fabricius, where severe cystic degeneration was commonly seen. In 51% of the pigeons confirmed as circovirus infected using EM, the histopathological detection of specific basophilic cytoplasmic inclusion bodies ( Figure 1) within medullary and cortical regions of bursal follicles allowed a clear diagnosis. Circovirus inclusions were round to ovoid shaped, sharply demarcated and frequently formed botryoid clusters. Occasionally inclusion bodies were detectable in spleen and thymus. Electron microscopy In negatively stained preparations, CoCV appeared as small naked icosahedral particles without recognizable surface structure ( Figure 2). The particle
size was about 14 to 16 nm when stained with phosphotungstic acid and 16 to 18 nm when stained with uranyl acetate. In our study, virus particles were detectable in 90% of bursal, 52% of thymic and 50% of splenic samples from circovirus infected pigeons. CoCV was only occasionally detected in the intestine, lung, trachea, kidney, liver or bone marrow. Diagnostic PCR A total of 41 samples were investigated by PCR for the presence of CoCV ( Table 1). When 12 lymphoreticular tissue samples from 11 animals that had been confirmed circovirus infected using EM were tested, a 336 bp amplicon was obtained from all of them ( data not shown). Sequencing of the PCR products revealed the expected CoCV-specific frag-
Figure 2. Transmission electron photomicrograph of CoCV particles negatively stained with phosphotungstic acid.
Pigeon circovirus infections 609
Figure 3. In situ hybridization. Bursal follicle showing intense CoCV nucleic acid labelling of medullary and cortical cells. Bar represents 50 mm.
ment. Another six lymphoreticular tissue samples from four animals, in which CoCV was not detected by EM, yielded positive PCR results. We investigated 15 samples of various organs from one pigeon, in which CoCV particles had been detected in the bursa, thymus, spleen and intestine using EM. The PCR assay detected CoCV in all samples: spleen, liver, kidney, lung, intestine, thymus, bursa, brain, gizzard, heart, muscle, skin, trachea and ovary ( Figure 3). DNA isolated from egg white and egg yolk of a freshly embryonated pigeon egg was negative. However, CoCV DNA was amplified from bursa, thymus or spleen from two embryos shortly before hatching, although not from their allantoic sacs. These results demonstrate that PCR is a powerful tool for CoCV detection. In situ hybridization Labelling was found mostly in the cytoplasm of infected cells and to a lesser extent in the nucleus. The amount of CoCV nucleic acid in tissues from the same pigeon was slightly variable. In most lymphoid tissues ( bursa of Fabricius, spleen and thymus), large to intermediate amounts of viral nucleic acid were detected primarily in the cytoplasm and/or the
nucleus of macrophages or histiocytic inflammatory cells, and reticular cells of the periellipsoidal cuffs of the spleen. Small round cells, which morphologically resembled lymphocytes, showed sporadic cytoplasmic labelling. Similarly, fibroblast-like cells located at the periphery of lymphoid follicles of the bursa of Fabricius had detectable viral nucleic acid within the cytoplasm. On the other hand, most of the cytoplasmic inclusion bodies observed in the follicles of the bursa had massive amounts of CoCV genome; only a few of them showed no staining. Distribution of the viral nucleic acid in the bursa of Fabricius was mainly follicular, both in the cortex and medulla ( Figure 4). The thymus had CoCV nucleic acid labelling mainly in the cortical areas, with lesser numbers of labelled cells in the thymic medulla; Hassall’s corpuscles and epithelial cells were not stained. The spleen had viral genome mainly in periellipsoidal cuffs and also within the cytoplasm of macrophages located in the red pulp and in subcapsular areas. In those cases with lymphoreticular hyperplasia of the spleen, labelling was located within macrophages and sporadic lymphocyte-like cells of the lymphoid follicles. Viral nucleic acid labelling in the small intestine of one pigeon was located within the cytoplasm of
Figure 4. Nested PCR with various organs from one CoCV-positive pigeon. The results of the second round of PCR are shown: Lane 1, no template ( negative control); 2, liver; 3, kidney; 4, lung; 5, intestine; 6, thymus; 7, bursa of Fabricius; 8, skeletal muscle; 9, heart; 10, skin; 11, brai; 12, trachea; 13, gizzard; 14, ovary; 15, spleen.
610 D. Soike et al.
macrophage-like cells ( also in intracytoplasmic inclusion bodies) in lymphocyte aggregates. No labelling was detected in the tissue of a pig infected with PCV2, nor in the tissue of a parrot infected with BFDV. A total number of 50 lymphoreticular tissue samples from 20 young pigeons were examined using both EM and ISH ( Table 2). EM detected circovirus in 37 samples, and CoCV nucleic acid was detectable in 36 samples by ISH. Corresponding results were obtained in 84% of the samples investigated. Concurrent infections A broad range of bacterial, viral, fungal and parasitic pigeon diseases were found as concurrent infections in 91% of the young pigeons infected with CoCV ( Table 3). Multiple concurrent infections were frequently observed. The detection of highly pathogenic agents, like Salmonella typhimurium, was more common in the circovirus-negative pigeons. On the other hand, agents of lower pathogenicity, such as Riemerellalike bacteria, were more common in circovirusinfected pigeons. Rotaviruses, rarely diagnosed in the pigeon, were detected only in three circovirusinfected birds in our study. Picorna virus-like particles, not previously detected in pigeons, were observed in another circovirus-positive bird. Discussion Circovirus infection in pigeons was demonstrated to be common throughout the study area. No other infection was detected with a comparable frequency in young diseased pigeons. Reliable diagnostic Table 3. Prevalence of infections in young pigeons positive or negative for CoCV as detected by EM
Infectious agent Salmonella typhimurium Escherichia coli Riemerella-like bacteria Pasteurella spp. Staphylococcus spp. Chlamydia psittacii Paramyxovirus Herpesvirus Adenovirus Reovirus Rotavirus Picornavirus-like particles Trichomonas gallinaea Coccidia spp. Candida spp. Aspergillus fumigatus a
CoCV-positive ( n = 83)
CoCV-negative ( n = 93)
17 14 15 3 3 7 11 7 7 2 3 1 17 13 4 2
47 12 4 4 3 3 12 2 10 2 – – 10 11 2 8
Based on the presence of a yellowish button-like coating on the pharyngeal mucosa.
tools are therefore needed. CoCV-infected pigeons did not show specific clinical signs, although a wasting syndrome was frequently reported. Circovirus-specific pathological changes were essentially restricted to the lymphoreticular tissue. Histopathological lesions did not always provide definitive evidence for CoCV infection, as specific inclusion bodies were found in only 51% of the pigeons confirmed to be circovirus-infected using EM. Several other agents cause damage to the lymphoreticular tissue and it may be difficult to differentiate lesions from age-related changes due to involution of the bursa and thymus. Negative-stained electron microscopic examination of lymphoreticular tissue, particularly the bursa of Fabricius, was shown to be a simple and fast method for circovirus detection. Since electron microscopy is not within reach for every diagnostic laboratory and PCR facilities are increasingly available, a PCR assay was developed for the detection of CoCV. The nested PCR used in this study detected 1.3 copies of a CoCV genome and is a highly sensitive diagnostic tool. Compared with ISH and EM, this PCR assay detected CoCV infection in larger numbers of pigeons. Analysis of a tissue collection from one diseased bird revealed CoCV throughout the tissues and organs. Further investigations are planned to evaluate the reliability of the nested PCR for CoCV detection in blood droplets dried on blotting or filter paper. A test like this would be helpful to examine the prevalence of CoCV among healthy pigeons. Detection of CoCV in embryonic tissue suggested vertical transmission of the agent and virus persistence in the adult pigeon. ISH results confirmed that histopathological changes of lymphoreticular tissue corresponded with the location of CoCV nucleic acid. A high correspondence between ISH and EM results indicated that ISH is also a reliable diagnostic tool. The grade of damage of the lymphoreticular tissue in CoCV-infected animals varied but a serious disturbance of immune system tissue was suggested in the majority of cases. Multiple infections and the detection of various agents of low or unknown pathogenicity indicated that acquired immunodeficiency may be associated with CoCV infection. Circovirus infection is therefore likely to have an impact on the course and severity of various concurrent infections. Experimental infections will be required to obtain definitive information on the pathogenicity of CoCV and its effects on the immune system. Cultivation of the virus remains a prerequisite for a deeper insight into virus properties and the development of immunodiagnostic methods. Acknowledgements The authors thank Beate Tutte and Roswitha Lorenz for technical assistance. Part of the study was financed by European Union grant QLRT-PL1999– 00307.
Pigeon circovirus infections 611
References Duchatel, J.P., Jauniaux, T., Vandersanden, F., Charlier, G., Coignoul, F. & Vindevogel, H. ( 1998). Premi`ere mise en e´ vidence en Belgique de particules ressemblant a` des circovirus chez le pigeon voyageur. Annales de M´edecine V´et´erinaire, 142, 425–428. Gerdes, G.H. ( 1993). Two very small viruses – a presumptive identification. Journal of the South African Veterinary Association, 64, 2. Hamel, A.L., Lin, L.L. & Nayar, G.P.S. ( 1998). Nucleotide sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs. Journal of Virology, 72, 5262–5267. Mankertz, A., Domingo, M., Folch, J.M., LeCann, P., Jestin, A., Segal´es, J., Chmielewicz, B., Plana-Duran, J. & Soike, D. ( 2000a). Characterisation of PCV-2 isolates from Spain, Germany and France. Virus Research, 66, 65–77. Mankertz, A., Hattermann, K., Ehlers, B. & Soike, D. ( 2000b). Cloning and sequencing of columbid circovirus ( CoCV), a new circovirus from pigeons. Archives of Virology, 145, 2469–2479. McNulty, M.S., Dale, J.L., Lukert, P., Mankertz, A., Randles, J.W. & Todd, D. ( 2000) Circoviridae, In M.H.V. van Regenmortel, C.M. Fauquet, D.H.L. Bishop, E.B. Carstens, M.K. Estes, S.M. Lemon, J. Maniloff, M.A. Mayo, D.J. McGeoch, C.R. Pringle & R.B. Wickner ( Eds.), Seventh Report of the International Committee on Taxonomy of Viruses ( pp. 299–303). New York: Academic Press. Meehan, B.M., McNeilly, F., Todd, D., Kennedy, S., Jewhurst, V.A., Ellis, J.A., Hassard, L.E., Clarc, E.G., Haines, D.M. & Allan, G.M. ( 1998). Characterization of novel circovirus DNAs associated with wasting syndromes in pigs. Journal of General Virology, 79, 2171–2179. Morozov, I., Sirinarumitr, T., Sorden, S.D., Halbur, P.G., Morgan, M.K., Yoon, K. & Paul, P.S. ( 1998). Detection of a novel strain of porcine circovirus in pigs with postweaning multisystemic wasting syndrome. Journal of Clinical Microbiology, 36, 2535–2541. Nayar, G.P.S., Hamel, A.L., Lin, L., Sachvie, C., Grudeski, E. & Spearman, G. ( 1999). Evidence for circovirus in cattle with respiratory disease and from aborted bovine fetuses. Canadian Veterinary Journal, 40, 277–278. Par´e, J.A., Brash, M.L., Hunter, D.B. & Hampson, R.J. ( 1999). Observations on pigeon circovirus infection in Ontario. Canadian Veterinary Journal, 40, 659–662. Schmidt, R.E. ( 1992). Circovirus in pigeons. Journal of the Association of Avian Veterinarians, 6, 204. Smyth, J.A. ( 1995). Circovirus in pigeons. Veterinary Record, 137, 623. Smyth, J.A. & Carroll, B.P. ( 1995). Circovirus infection in European racing pigeons. Veterinary Record, 136, 173–174. Soike, D. ( 1997). Circovirusinfektion bei Tauben. Tier¨arztliche Praxis, 25, 52–54. Soike, D., K¨o hler, B. & Albrecht, K. ( 1999). A circovirus-like infection in geese related to a runting syndrome. Avian Pathology, 28, 199–202. Todd, D. ( 2000). Circoviruses: immunosuppressive threats to avian species: a review. Avian Pathology, 29, 373–394. Todd., D., Weston, J., Ball, N.W., Borghmans, B.J., Smyth, J.A., Gelmini, L. & Lavazza, A. ( 2001). Nucleotide sequence-based identification of a novel circovirus of canaries. Avian Pathology, 30( 4), 321–325. Twentyman, C.M., Alley, M.R., Meers, J., Cooke, M.M. & Duignan, P.J. ( 1999). Circovirus-like infection in a southern black-backed gull ( Larus dominicanus). Avian Pathology, 28, 513–516. Woods, L.W., Latimer, K.S., Barr, B.C., Niagro, F.D., Campagnoli, R.P., Nordhausen, R.W. & Castro, A.E. ( 1993). Circovirus-like infection in a pigeon. Journal of Veterinary Diagnostic Investigation, 5, 609–612.
Woods, L.W., Latimer, K.S., Niagro, F.D., Riddell, C., Crowley, A.M., Anderson, M.L., Daft, B.M., Moore, J.D., Campagnoli, R.P. & Nordhausen, R.W. ( 1994). A retrospective study of circovirus infection in pigeons: nine cases ( 1986–1993) . Journal of Veterinary Diagnostic Investigation, 6, 156–164. ´ RESUM E´ Etude diagnostique sur l’infection a` Circovirus des colombid´es Une e´ tude syst´ematique a e´ t´e entreprise pour connaˆõ tre la fr´equence de l’infection a` Circovirus des colombid´es ( CoCV), chez des jeunes pigeons malades soumis a` autopsie et e´ galement chez des pigeons sains. Les m´ethodes de diagnostic disponibles ont e´t´e compar´ees. A partir de 176 jeunes pigeons malades examin´es, l’infection a` CoCV a e´ t´e mise en e´ vidence chez 83 d’entre eux, en utilisant la coloration n´egative en microscopie e´lectronique. Les examens en histopathologie n’ont permis de diagnostiquer l’infection que chez 42 pigeons seulement. Alors, un test d’amplification en chaˆõ ne par polym´erase ( PCR), ainsi qu’un test d’hybridation in situ ( ISH) ont e´ t´e d´evelopp´es. Le CoCV est de loin l’agent infectieux le plus souvent d´etect´e chez les pigeons malades. Les pigeons infect´es ont pr´esent´e une grande diversit´e d’infections intercurrentes. Les observations pathologiques sugg`erent que le CoCV a un effet immunod´epresseur.
ZUSAMMENFASSUNG Eine diagnostische Studie u¨ ber die Tauben-Circovirusinfektion Es wurde eine systematische Studie durchgef¨uhrt, um die H¨aufigkeit der Infektion mit columbidem Circovirus ( CoCV) bei zur Sektion eingelieferten kranken Jungtauben und dessen Bedeutung f¨u r die Taubengesundheit zu untersuchen. Die vorhandenen diagnostischen Methoden wurden verglichen. Unter 176 untersuchten kranken Jungtauben wurde die CoCV-Infektion mit der Negativkontrast-Elektronenmikroskopie in 83 F¨allen nachgewiesen. Die histologische Untersuchung erlaubte bei nur 42 Tauben eine eindeutige Diagnose. Deshalb wurden sowohl ein Polymerase-Kettenreaktion ( PCR)-Test als auch ein In-situ-Hybridisierung ( ISH) -Test als zus¨atzliche diagnostische Hilfsmittel entwickelt. CoCV ist der weitaus am h¨aufigsten nachgewiesene Infektionserreger bei erkrankten Jungtauben. Infizierte Tauben haben ein breites Spektrum gleichzeitiger Infektionen. Die pathologischen Befunde lassen auf eine immunsuppressive Wirkung von CoCV schließen.
RESUMEN Estudio sobre el diagn´ostico de la infecci´o n por circovirus de paloma Se llev´o a cabo un estudio sistem´atico para determinar, por un lado, la frecuencia de la infecci´on por circovirus de la paloma ( CoCV) en palomas j´o venes enfermas remitidas para necropsia y, por otro lado, la relevancia de este agente v´õ rico en la salud de las palomas. Tambi´en se compararon los m´etodos diagn´osticos existentes. De 176 palomas j´o venes enfermas examinadas, se detect´o infecci´o n por CoCV en 83 casos mediante microscop´õ a electro´ nica utilizando contraste negativo. El examen histopatol´o gico u´ nicamente permiti´o un diagn´o stico claro en 42 palomas. En consecuencia, se desarrollaron, como t´ecnicas de diagn´ostico complementarias, la t´ecnica de reacci o´ n en cadena de la polimerasa ( PCR), as´õ como el test de hibridaci´o n in situ ( ISH). El CoCV es el agente infeccioso que se detecta con m´as frecuencia en palomas j´ovenes enfermas. Las palomas infectadas mostraron adem´as un amplio rango de infecciones concomitantes. Los hallazgos histopatol´o gicos sugieren un efecto inmunosupresivo del CoCV.
