The pigeon circovirus evolution, epidemiology and interaction with the host immune system under One Loft Race rearing conditions.

This study was aimed to investigate the frequency of PiCV recombination, the kinetics of PiCV viremia and shedding and the correlation between viral replication and host immune response in young pigeons subclinically infected with various PiCV variants and kept under conditions mimicking the OLR system. Fifteen racing pigeons originating from five breeding facilities were housed together for six weeks. Blood and cloacal swab samples were collected from birds every seven days to recover complete PiCV genomes and determine PiCV genetic diversity and recombination dynamics, as well as to assess virus shedding rate, level of viremia, expression of selected genes and level of anti-PiCV antibodies. Three hundred and eighty-eight complete PiCV genomes were obtained and thirteen genotypes were distinguished. Twenty-five recombination events were detected. Recombinants emerged during the first three weeks of the experiment which was consistent with the peak level of viremia and viral shedding. A further decrease in viremia and shedding partially corresponded with IFN-γ and MX1 gene expression and antibody dynamics. Considering the role of OLR pigeon rearing system in spreading infectious agents and allowing their recombination, it would be reasonable to reflect on the relevance of pigeon racing from both an animal welfare and epidemiological perspective.

Investigating pigeon circovirus infection in a pigeon farm: molecular detection, phylogenetic analysis and complete genome analysis.

BACKGROUND: Pigeon circovirus infections in pigeons (Columba livia domestica) have been reported worldwide. Pigeons should be PiCV-free when utilized as qualified experimental animals. However, pigeons can be freely purchased as experimental animals without any clear guidelines to follow. Herein, we investigated the status quo of PiCV infections on a pigeon farm in Beijing, China, which provides pigeons for experimental use. RESULTS: PiCV infection was verified in at least three types of tissues in all forty pigeons tested. A total of 29 full-length genomes were obtained and deposited in GenBank. The whole genome sequence comparison among the 29 identified PiCV strains revealed nucleotide homologies of 85.8-100%, and these sequences exhibited nucleotide homologies of 82.7-98.9% as compared with those of the reference sequences. The cap gene displayed genetic diversity, with a wide range of amino acid homologies ranging from 64.5% to 100%. Phylogenetic analysis of the 29 full-genome sequences revealed that the PiCV strains in this study could be further divided into four clades: A (17.2%), B (10.4%), C (37.9%) and D (34.5%). Thirteen recombination events were also detected in 18 out of the 29 PiCV genomes obtained in this study. Phylogenetic research using the rep and cap genes verified the recombination events, which occurred between clades A/F, A/B, C/D, and B/D among the 18 PiCV strains studied. CONCLUSIONS: In conclusion, PiCV infection, which is highly genetically varied, is extremely widespread on pigeon farms in Beijing. These findings indicate that if pigeons are to be used as experimental animals, it is necessary to evaluate the impact of PiCV infection on the results.

Australasian Pigeon Circoviruses Demonstrate Natural Spillover Infection.

Pigeon circovirus (PiCV) is considered to be genetically diverse, with a relatively small circular single-stranded DNA genome of 2 kb that encodes for a capsid protein (Cap) and a replication initiator protein (Rep). Australasia is known to be the origin of diverse species of the Order Columbiformes, but limited data on the PiCV genome sequence has hindered phylogeographic studies in this species. To fill this gap, this study was conducted to investigate PiCV in 118 characteristic samples from different birds across Australia using PCR and sequencing. Eighteen partial PiCV Rep sequences and one complete PiCV genome sequence were recovered from reservoir and aberrant hosts. Phylogenetic analyses revealed that PiCV circulating in Australia was scattered across three different subclades. Importantly, one subclade dominated within the PiCV sequenced from Australia and Poland, whereas other PiCV sequenced in this study were more closely related to the PiCV sequenced from China, USA and Japan. In addition, PiCV Rep sequences obtained from clinically affected plumed whistling duck, blue billed duck and Australian magpie demonstrated natural spillover of PiCV unveiled host generalist characteristics of the pigeon circovirus. These findings indicate that PiCV genomes circulating in Australia lack host adapted population structure but demonstrate natural spillover infection.

Lesions and viral loads in racing pigeons naturally coinfected with pigeon circovirus and columbid alphaherpesvirus 1 in Australia.

Columbid alphaherpesvirus 1 (CoHV1) is associated with oral or upper respiratory tract lesions, encephalitis, and occasional fatal systemic disease in naive or immunosuppressed pigeons. Clinical disease is often reported with CoHV1 and coinfecting viruses, including pigeon circovirus (PiCV), which may cause host immunosuppression and augment lesion development. A natural outbreak of CoHV1 and PiCV coinfection occurred in a flock of 60 racing rock pigeons (Columba livia), in which 4 pigeons succumbed within 7 d of clinical onset. Lesions included suppurative stomatitis, pharyngitis, cloacitis, meningitis, and tympanitis, with eosinophilic intranuclear inclusion bodies consistent with herpesviral infection. In addition, large numbers of botryoid intracytoplasmic inclusion bodies were present in the skin, oral mucosa, and bursa of Fabricius, suggestive of circoviral infection, which was confirmed by immunohistochemistry. The concurrent viral load of CoHV1 and PiCV was high in liver, oropharynx, and bursa of Fabricius. We found PiCV in oro-cloacal swabs from 44 of 46 additional birds of variable clinical status, PiCV alone in 23 birds, and coinfection with CoHV1 in 21 birds. Viral copy numbers were significantly higher (p < 0.0001) for both viruses in clinically affected pigeons than in subclinical qPCR-positive birds. The CoHV1-induced lesions might have been exacerbated by concomitant PiCV infection.

Pigeon Circovirus over Three Decades of Research: Bibliometrics, Scoping Review, and Perspectives.

The pigeon circovirus (PiCV), first described in the literature in the early 1990s, is considered one of the most important infectious agents affecting pigeon health. Thirty years after its discovery, the current review has employed bibliometric strategies to map the entire accessible PiCV-related research corpus with the aim of understanding its present research landscape, particularly in consideration of its historical context. Subsequently, developments, current knowledge, and important updates were provided. Additionally, this review also provides a textual analysis examining the relationship between PiCV and the young pigeon disease syndrome (YPDS), as described and propagated in the literature. Our examination revealed that usages of the term 'YPDS' in the literature are characterizations that are diverse in range, and neither standard nor equivalent. Guided by our understanding of the PiCV research corpus, a conceptualization of PiCV diseases was also presented in this review. Proposed definitions and diagnostic criteria for PiCV subclinical infection (PiCV-SI) and PiCV systemic disease (PiCV-SD) were also provided. Lastly, knowledge gaps and open research questions relevant to future PiCV-related studies were identified and discussed.

Development and applications of a TaqMan based quantitative real-time PCR for the rapid detection of Pigeon circovirus (PiCV).

TaqMan probe based quantitative polymerase reaction (TaqMan qPCR) is a robust and reliable technique for detecting and quantifying target DNA copies. Quantitative molecular diagnosis of genetically diverse single stranded DNA (ssDNA) virus such as Pigeon circovirus (PiCV) can be challenging owing to difficulties in primer binding or low abundance of template DNA copies in clinical specimens. Several methods have been described for the detection of PiCV, being qPCR the most simple and reliable. As far as is known, two qPCR systems described until now are based on SYBR green. This study reports development and validation of a highly sensitive TaqMan qPCR targeted to Rep for the detection of highly diverse PiCV in pigeon samples with excellent reproducibility, specificity, and sensitivity. The limit of detection was determined as low as 2 (two) plasmid copies. Estimations of 100 % specificity and 100 % sensitivity were obtained based on the qPCR results with panel of 60 samples (known PiCV positive, n = 30; known PiCV negative, n = 20; samples positive to Beak and feather disease virus (BFDV), n = 5 and samples positive to canine circovirus, n = 5). Co-efficient of variation (CV) for Ct values ranged between 0.27 % and 0.78 % in the same assay and 1.84-2.87 % in different assays.

Molecular detection and phylogenetic analysis of pigeon circovirus from racing pigeons in Northern China.

BACKGROUND: Pigeon circovirus (PiCV) infections in pigeons (Columba livia) have been reported worldwide. Currently, pigeon racing is becoming increasingly popular and considered to be a national sport in China, and even, the greatest competitions of racing pigeons are taking place in China. However, there are still no epidemiologic data regarding PiCV infections among racing pigeons in China. The purpose of our study was to provide information of prevalence, genetic variation and evolution of PiCV from racing pigeons in China. RESULTS: To trace the prevalence, genetic variation and evolution of PiCV in sick and healthy racing pigeons, 622 samples were collected from 11 provinces or municipalities in China from 2016 to 2019. The results showed that the positive rate of PiCV was 19.3% (120/622) at the sample level and 59.0% (23/39) at the club level, thus suggesting that the virus was prevalent in Chinese racing pigeons. A sequence analysis revealed that the cap genes of the PiCV strains identified in our study displayed a high genetic diversity and shared nucleotide homologies of 71.9%-100% and amino acid homologies of 71.7%-100%. 28 and 36 unique amino acid substitutions were observed in the Cap and Rep proteins derived from our PiCV strains, respectively. A cladogram representation of PiCV strains phylogeny based on 90 cap gene sequences showed that the strains in this study could be further divided into seven clades (A, B, C, E, G, H, and I) and some of them were closely related to worldwide strains from different types of pigeons. A large number of recombination events (31 events) were also detected in the PiCV genomes from Chinese racing pigeons. CONCLUSIONS: These findings indicate that PiCV strains circulating in China exhibit a high genetic diversity and also contribute to information of prevalence, genetic variation and evolution of PiCV from racing pigeons in China.

First detection and molecular characterisation of a pigeon aviadenovirus A and pigeon circovirus co-infection associated with Young Pigeon Disease Syndrome (YPDS) in Turkish pigeons (Columba livia domestica).

Pigeon aviadenovirus A and Pigeon circovirus are both DNA viruses, infect and cause severe clinical diseases in pigeons. These viruses are associated with an immunosuppression syndrome similar to 'Young Pigeon Disease Syndrome' (YPDS). This study reports the identification of a natural co-infection, with severe clinical signs (crop vomiting, watery diarrhoea, anorexia and sudden death) of Pigeon aviadenovirus A and Pigeon circovirus in a breeding pigeon flock in Central Anatolia, Turkey. Both viruses were isolated from pigeons pooled internal organs using primary chicken embryo kidney cell cultures (CEKC) and specific pathogen-free (SPF) embryonated chicken eggs. Also, both viruses were identified by PCR amplification followed by Sanger sequencing whereas histopathological examination showed degenerated hepatocytes with basophilic intranuclear viral inclusions. As known, both viruses typically have similar transmission characteristics and common clinical manifestations; however, co-infection may exacerbate the disease with devastating outcomes. This is the first report of its kind in Turkey for those viruses and is essential for the protection against these kinds of infections in pigeons.

Investigation of Lethal Concurrent Outbreak of Chlamydiosis and Pigeon Circovirus in a Zoo.

During the spring, an outbreak of sudden death involving 58 birds occurred in a zoo. Histopathological examinations revealed variable numbers of intracytoplasmic basophilic microorganisms in the macrophages, hepatocytes, and renal epithelium of most birds, along with occasional botryoid intracytoplasmic inclusion bodies within histiocytes in the bursa of Fabricius. Based on the results of histopathological examinations, immunohistochemical staining, transmission electron microscopy, and polymerase chain reactions, genotype B Chlamydia psittaci infection concurrent with pigeon circovirus (PiCV) was diagnosed. A retrospective survey, including two years before the outbreak and the outbreak year, of C. psittaci and PiCV infections of dead birds in the aviaries, revealed that the outbreak was an independent episode. The findings of this study indicate that concurrent infection with C. psittaci and PiCV might lead to lethal outbreaks of chlamydiosis, particularly Streptopelia orientalis. In addition, persistently monitoring both pathogens and identifying potential PiCV carriers or transmitters might also help prevent lethal disease outbreaks.

A Pilot Study Investigating the Dynamics of Pigeon Circovirus Recombination in Domesticated Pigeons Housed in a Single Loft.

Pigeon circovirus (PiCV) infects pigeon populations worldwide and has been associated with immunosuppression in younger pigeons. Recombination is a common mechanism of evolution that has previously been shown in various members of the Circoviridae family, including PiCV. In this study, three groups of pigeons acquired from separate lofts were screened for PiCV, and their genome sequence was determined. Following this, they were housed in a single loft for 22 days, during which blood and cloacal swab samples were taken. From these blood and cloacal swabs, PiCV genomes were determined with the aim to study the spread and recombination dynamics of PiCV in the birds. Genome sequences of PiCV were determined from seven pigeons (seven tested PiCV positive) before they were housed together in a loft (n = 58 sequences) and thereafter from the ten pigeons from blood and cloacal swabs (n = 120). These 178 PiCV genome sequences represent seven genotypes (98% pairwise identity genotype demarcation), and they share >88% genome-wide pairwise identity. Recombination analysis revealed 13 recombination events, and a recombination hotspot spanning the 3' prime region, the replication-associated protein (rep) gene and the intergenic region. A cold spot in the capsid protein-coding region of the genome was also identified. The majority of the recombinant regions were identified in the rep coding region. This study provides insights into the evolutionary dynamics of PiCV in pigeons kept under closed rearing systems.

Immunogenicity and Protective Activity of Pigeon Circovirus Recombinant Capsid Protein Virus-Like Particles (PiCV rCap-VLPs) in Pigeons (Columba livia) Experimentally Infected with PiCV.

Pigeon circovirus (PiCV) is the most recurrent virus diagnosed in pigeons and is among the major causative agents of young pigeon disease syndrome (YPDS). Due to the lack of an established laboratory protocol for PiCV cultivation, development of prophylaxis is hampered. Alternatively, virus-like particles (VLPs), which closely resemble native viruses but lack the viral genetic material, can be generated using a wide range of expression systems and are shown to have strong immunogenicity. Therefore, the use of VLPs provides a promising prospect for vaccine development. In this study, transfected human embryonic kidney (HEK-293) cells, a mammalian expression system, were used to express the PiCV capsid protein (Cap), which is a major component of PiCV and believed to contain antibody epitopes, to obtain self-assembled VLPs. The VLPs were observed to have a spherical morphology with diameters ranging from 12 to 26 nm. Subcutaneous immunization of pigeons with 100 µg PiCV rCap-VLPs supplemented with water-in-oil-in-water (W/O/W) adjuvant induced specific antibodies against PiCV. Observations of the cytokine expression and T-cell proliferation levels in spleen samples showed significantly higher T-cell proliferation and IFN- γ expression in pigeons immunized with VLPs compared to the controls (p < 0.05). Experimentally infected pigeons that were vaccinated with VLPs also showed no detectable viral titer. The results of the current study demonstrated the potential use of PiCV rCap-VLPs as an effective vaccine candidate against PiCV.

Probiotic supplementation containing Bacillus velezensis enhances expression of immune regulatory genes against pigeon circovirus in pigeons (Columba livia).

AIMS: In this study, we aimed to isolate and evaluate the efficacy of Bacillus velezensis as a probiotic and to assess its activity towards pigeons infected with pigeon circovirus (PiCV). METHODS AND RESULTS: Bacillus velezensis, isolated from pigeon faeces, was orally administered to pigeons for 60 days. After pigeons were challenged with PiCV, the PiCV viral load and expression of indicator genes for innate immunity were detected in spleen tissue and faeces of pigeons. Bacillus velezensis significantly reduced the PiCV viral load in the faeces and spleen of pigeons 5 days post-challenge (dpc). The mRNA expression levels of treated pigeons showed that interferon-gamma (IFN-γ), myxovirus resistance 1 (Mx1), and signal transducers and activators of transcription 1 (STAT1) genes were upregulated, whereas no expression of interleukin-4 (IL-4) was detected. Moreover, toll-like receptor 2 (TLR2) and 4 (TLR4) were significantly upregulated in probiotic-treated pigeons (P < 0·05). CONCLUSIONS: This is the first report showing that probiotic supplementation can effectively enhance the T-helper type 1 immune response and decrease the PiCV viral loads in pigeons. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proposes that the administration of a probiotic strain, B. velezensis, to pigeons can protect against PiCV infection.

The Clinical Infection with Pigeon Circovirus (PiCV) Leads to Lymphocyte B Apoptosis But Has No Effect on Lymphocyte T Subpopulation.

The pathology of pigeon circovirus (PiCV) is still unknown, but it is regarded as an immunosuppressant. This study aimed to find a correlation between PiCV natural infection and immunosuppression. The study was conducted with 56 pigeons divided into the following groups: PiCV-positive but showing (group S) or not (group I) non-specific clinical symptoms and asymptomatic pigeons negative for PiCV (group H). The percentage and apoptosis of T CD3+ and B IgM+ splenocytes; the expression of CD4, CD8, and IFN-γ genes in splenic mononuclear cells; the number of PiCV viral loads in the bursa of Fabricius; and the level of anti-PiCV antibodies were analyzed. The results showed that the percentage of B IgM+ cells was almost two-fold lower in group S than in group H, and that ca. 20% of the lymphocytes were apoptotic. No increased apoptosis was detected in TCD3+ subpopulation. The PiCV viral loads were approximately one thousand and ten thousand times higher in group S than in groups I and H, respectively. Our results indicate a possible correlation between the number of PiCV viral loads and severity of PiCV infection and confirm that PiCV infection leads to the suppression of humoral immunity by inducing B lymphocyte apoptosis.

Influence of pigeon interferon alpha (PiIFN-α) on pigeon circovirus (PiCV) replication and cytokine expression in Columba livia.

Pigeon circovirus (PiCV) is the most diagnosed virus in pigeons (Columba livia) and have been studied and reported globally. PiCV infections can lead to immunosuppression and pigeons infected with PiCV can result to lymphocyte apoptosis and atrophy of immune organs. Young pigeon disease syndrome (YPDS) is a complex disease and believed that PiCV could be one of the agents leading to this syndrome. An effective treatment regimen is needed to control the spread of PiCV in pigeons. In this study pigeon interferon alpha (PiIFN-α) was cloned and expressed and its antiviral effects were tested against fowl adenovirus type 4 (FAdV-4) in vitro and PiCV in vivo. No detectable levels of FAdV-4 viral genome in LMH cells stimulated with 300 µg/mL PiIFN-α were found. Additionally, PiIFN-α was stable at different temperature and pH for 4 h, and no reduction in antiviral activity was observed in untreated and treated cells. In pigeons naturally and experimentally infected by PiCV, no detectable levels of PiCV virus titers were found after treatment with PiIFN-α. Cytokine and ISG expression levels in liver and spleen samples were detected and IFN-γ and Mx1 genes were dominantly up-regulated following PiIFN-α treatment (p < 0.05). This study demonstrated that PiCV can be inhibited by administration of PiIFN-α and PiFN-α can be used as a therapeutic approach to prevent the spread of PiCV in pigeons.

Pigeon circoviruses from feral pigeons in Australia demonstrate extensive recombination and genetic admixture with other circoviruses.

Like other avian circovirus species, Pigeon circovirus (PiCV) is known to be genetically diverse with a relatively small circular single-stranded DNA genome of 2 kb that encodes for a capsid protein (Cap) and a replication initiator protein (Rep). Recent paleoviral evidence hints towards a probable Gondwanan origin of avian circoviruses, paralleling the evolution and dispersal of their hosts. Limited availability of PiCV genome sequence data in Australia has hindered phylogeographic studies in this species, so we screened clinically normal rock doves (Columba livia) in regional New South Wales, and demonstrated a high prevalence (76%) of PiCV infection by PCR. We also recovered 12 complete novel PiCV genomes and phylogenetic analyses revealed that PiCV circulating in Australian feral pigeons formed two strongly supported monophyletic clades. One clade resided with PiCV genomes from Poland, Australia, United Kingdom, Belgium, China, and Japan, and another basal clade was more closely related to PiCV genomes from Poland. A novel more distantly-related PiCV rep gene formed a solitary clade with weak posterior support. So we further analysed all selected partial rep gene sequences to demonstrate a likely naturally occurring spillover infection from a passerine circovirus candidate. The findings suggest that there is a high degree of genetic variation within PiCV in Columbiformes with potential greater admixture between avian circoviruses within Australia than previously known. RESEARCH HIGHLIGHTS Confirmed high prevalence rate of PiCV circulating in Australian wild pigeons. Highlighted extensive recombination events within Australian PiCV. Demonstrated a likely naturally occurring spillover infection from a passerine circovirus candidate.

Immunogenicity of Pigeon Circovirus Recombinant Capsid Protein in Pigeons.

Pigeon circovirus (PiCV) is the most frequently diagnosed virus in pigeons and is thought to be one of the causative factors of a complex disease called the young pigeon disease syndrome (YPDS). The development of a vaccine against this virus could be a strategy for YPDS control. Since laboratory culture of PiCV is impossible, its recombinant capsid protein (rCP) can be considered as a potential antigen candidate in sub-unit vaccines. The aim of this basic research was to evaluate the immune response of pigeons to PiCV rCP. Sixty six-week-old carrier pigeons were divided into two groups (experimental immunized with PiCV rCP mixed with an adjuvant, and control immunized with an adjuvant only), and immunized twice in a 21-day interval. On the day of immunization and on two, 23, 39, and 46 days post first immunization (dpv), samples of blood, spleen, and bursa of Fabricius were collected from six birds from each group to examine anti-PiCV rCP IgY, anti-PiCV rCP IgY-secreting B cells (SBC), IFN-γ gene expression, and percentage of T CD3⁺, CD4⁺, CD8⁺, and B IgM⁺ lymphocytes. The results indicated a correct immune response to PiCV rCP both in humoral and cell-mediated immunity, which was manifested by seroconversion since 23 dpv, by a significantly higher anti-PiCV rCP IgY-SBC number on two and 23 dpv, and significantly higher IFN-γ gene expression since two dpv. There were no significant differences or trends noted between particular T and B lymphocyte subpopulations. To conclude, PiCV rCP may be deemed immunogenic and could be considered as an antigen candidate in sub-unit vaccines against PiCV infections in pigeons.

The epidemiology, molecular characterization and clinical pathology of circovirus infections in pigeons - current knowledge.

The first cases of circovirus infections in pigeons were documented less than 25 years ago. Since then, circovirus infections have been reported on nearly all continents. The specificity of pigeon breeding defies biosecurity principles, which could be the reason for the high prevalence of PiCV infections. PiCV infections in pigeons lead to atrophy of immune system organs and lymphocyte apoptosis. Infected birds could be more susceptible to infections of the respiratory and digestive tract. PiCV has been associated with the young pigeon disease syndrome (YPDS). PiCVs are characterized by high levels of genetic diversity due to frequent point mutations, recombination processes in the PiCV genome and positive selection. Genetic recombinations and positive selection play the key role in the evolution of PiCV. A protocol for culturing PiCV under laboratory conditions has not yet been developed, and traditional vaccines against the infection are not available. Recombinant capsid proteins for detecting anti-PiCV antibodies have been obtained, and these antigens can be used in the production of diagnostic tests and subunit vaccines against PiCV infections. However, YPDS has complex etiology, and it remains unknown whether immunization against PiCV alone will contribute to effective control of YPDS.

Genome sequence characterization of pigeon circoviruses in China.

Pigeon circovirus (PiCV) was detected by PCR in pigeons from China. Altogether, 48 out of 244 pigeons tested positive for PiCV (positive rate, 19.67%), suggesting that the virus was prevalent in China. From the 48 PiCV-positive samples, about 2040bp complete genome fragments were obtained by full length genome amplification and sequenced with a next-generation sequencing platform. Characteristics of the ORFs from different PiCV strains tested in this study were analyzed. Several insertion, deletion or substitutions were discovered during the analysis of the nucleotide sequence compared with sequences reported previously. In phylogenetic tree analysis, 48 sequences isolated in this study could be further divided into five clades (A, B, C, D, and F), clade E includes reference sequences only. Two major groups were found in the six clades, distinguished by ATA and ATG initiation codons. Most of the viruses isolated in the study were in the ATG group, with fewer in the ATA branch.

Pigeon circovirus infection in disqualified racing pigeons from Taiwan.

Pigeons (Columba livia) infected with pigeon circovirus (PiCV) have been reported worldwide. The present study diagnosed PiCV infection in tissue samples of disqualified racing pigeons in Taiwan, using molecular and microscopy diagnostics. Among the 164 dead pigeons examined, 96.95% (159/164) tested positive for PiCV. Severe histopathological lesions, with characteristic inclusions, were observed in various organs of the PiCV-infected pigeons. Multiglobular basophilic intranuclear and intracytoplasmic inclusion bodies were found in the bursa of Fabricius and non-lymphoid tissues. The present study identified, for the first time, the presence of inclusion bodies in the thyroid gland, oesophagus, gizzard, and in the third eyelid of circovirus-infected pigeons. The presence of inclusion bodies in the third eyelid and mucosa of the gizzard was confirmed by transmission electron microscopy. A high detection rate of PiCV and some severe lesions evident in disqualified racing pigeons, as well as PiCV sequences in this study were highly similar with those detected in European countries suggesting an epidemiological association possibly due to imported pigeons.

Application of pigeon circovirus recombinant capsid protein for detecting anti-PiCV antibodies in the sera of asymptomatic domestic pigeons and the potential use of a combination of serological and molecular tests for controlling circovirus infections in pigeon breeding flocks.

The aim of this study was to evaluate the serologic status of domestic pigeons not infected and asymptomatically infected with the pigeon circovirus (PiCV) with the use of an enzyme-linked assay based on PiCV recombinant capsid protein as a plate antigen. Recombinant PiCV capsid protein was produced by transforming E. coli BL21 (DE3) Rosetta colonies with expression plasmids.Blood samples and cloacal swabs were collected from 171 asymptomatic pigeons. The birds were divided into two groups (infected and not infected with PiCV) based on the results of Sybr Green real time PCR screening for the presence of PiCV genetic material. Approximately 70% of the pigeons tested positive for anti-PiCV antibodies regardless of their infection status. Antibody levels, the coefficient of variation and standard deviation were significantly higher in the group of infected pigeons.The results indicate that ELISA is a highly useful test that complements molecular methods in evaluations of PiCV infection status in domestic pigeons. The spread of pigeon circovirus infections can be controlled by keeping breeding flocks free of PiCV, which can only be achieved by subjecting birds to real time PCR and serological tests.