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)

Abstract 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.


INTRODUCTION
Viral infections of pigeons, particularly under the age of 1 year old, are associated with high morbidity and mortality. It has been reported that Pigeon aviadenovirus A (PiAdV-A) (De Herdt et al., 1995;Duchatel et al., 2000) and pigeon circovirus (PiCV) (Todd, 2000) are associated with an immunosuppression syndrome similar to 'Young Pigeon Disease Syndrome' (YPDS). A multifactorial infection involves other crucial influencers like stressful conditions and other opportunistic pathogens, especially Escherichia (E.) coli (Raue et al., 2005). Flock morbidity and mortality for YPDS occur in juvenile pigeons only with up to 20 %; a rate considered lower when compared to other fatal infections in young and adult domestic pigeons like rotavirus A (RVA)-associated disease, in which flock morbidity is usually high and can reach up 100% (Schmidt et al., 2021).

PiAdV-A is a member of the genus Aviadenovirus within the family
Adenoviridae. To date, the Aviadenovirus genus is made up of 15 different species capable of infecting many avian species (Harrach et al., 2011;Marek et al., 2014b;Raue et al., 2002). Pigeon circovirus is a member of the Circovirus genus, family Circoviridae and within this genus, 11 different circoviruses could affect birds (Mankertz et al., 2000;Rosario et al., 2017).
Adenovirus infection of a pigeon was first reported in 1976 (McFerran et al., 1976a). Pigeons infected with PiAdV have subsequently been identified in many parts of the world (Vereecken et al., 1998). Although pigeons of any age can be infected, young pigeons under 1 year of age are particularly severely affected by PiAdV-A, showing acute watery diarrhoea, vomiting and anorexia. PiAdV-B affects pigeons of all ages and is characterised by sudden death and intensive hepatic necrosis (De Herdt et al. 1995;Duchatel et al. 2000;Vereecken et al., 1998).
The annual pigeon mortality rate is very high, with approximately 30% due to PiAdV, but in some cases, it can reach 100% in pigeon lofts with necrotising hepatitis infections (Vereecken et al., 1998).
PiCV was first diagnosed in Canada in 1986, and it is now considered to have a widespread distribution across the world (Woods et al., 1993). Similar to PiAdV-A, it predominantly causes disease in younger pigeons, mainly between two months and one year of age, with infection causing a broad spectrum of non-specific clinical signs including lethargy, weight loss, respiratory distress and diarrhoea (Pare et al., 1999;Takase et al., 1990;Tavernier et al., 2000;Todd, 2000;Woods et al., 1994).
Until recently, it was unclear what effect many aviadenoviruses and circoviruses had on pigeons, but now the impact of those viruses such as the fowl aviadenoviruses (FAdV) is well recognised recently due to their role in the multifactorial infection of YPDS as most of the previous studies pointed to the isolation of both viruses from different samples (Hess, 2013;Stenzel et al., 2012). These FAdV strains can cause gizzard erosion (GE), hydropericardium syndrome (HS) and severe liver damage leading to inclusion body hepatitis (IBH) and serotypes 2, 4, 5, 6, 8, 10 and 12 have been isolated from both diseased and healthy pigeons (Goryo et al., 1988;Hess et al., 1998a;Hess et al., 1998b;McFerran et al., 1976a). To increase our understanding of the aforementioned viruses' role in infecting these birds, we report here the isolation and analysis of both PiAdV and PiCV from a co-infection in pigeons in Central Anatolia, Turkey.

History, gross findings and sampling
A pigeon flock (n = 45, 4-5 months old domestic pigeons) had a history of increased mortality (20-25%). According to the pigeons' owner, the first sign was inappetence and vomiting. This was followed by dark green watery diarrhoea that continued for 2-5 days, with many pigeons died during this time. A total of five dead pigeons were collected after a period of chronic weight loss. Two out of the five birds had severe intestinal ascariasis and mucosal oedema. Systematically sampled tissue samples were fixated in 10% neutral formalin for pathology, and liver, kidney, spleen, gut and pancreas were collected under aseptic conditions for virus isolation and identification.
The organs were homogenised, followed by centrifugation (4000 rpm for 10 min). The supernatants were then collected for screening by PCR and virus isolation.

Isolation in SPF embryonated chicken eggs
Supernatants of pigeons pooled organs that were positive by PCR for PiAdV-A and PiCV were first filtered (0.22 μm microfilter) and then inoculated into the chorioallantoic cavity of 10-day-old specific pathogen-free (SPF) embryonated chicken eggs and the yolk sac of 6-day-old SPF eggs to observe and compare the viruses propagation according to the protocol of the Villegas Laboratory Manual (Villegas, 2006). The eggs were incubated at 37 • C for 5 or 10 days according to the inoculation route, respectively. The inoculated eggs were examined on a daily basis until embryos stopped moving and were presumed dead under ovoscope light. At this point, the allantoic fluid was collected and inoculated into fresh SPF eggs following the same procedure as before.
This was repeated until each supernatant was passaged through five eggs.

Isolation in cell culture
Supernatants to be used for inoculation of cell cultures were prepared from pooled organs and screened by PCR as described in the previous section. Primary chicken embryo fibroblast (CEF) and chicken embryo kidney cell (CEKC) cultures were prepared from 10-day-old and 18-day-old SPF embryonated chicken eggs according to the protocol of the Villegas Laboratory Manual, respectively (Villegas, 2006

Necropsy and histopathological examinations
After necropsy, multiple tissue samples were fixed in 10% neutral formalin (pH 7.4) and processed according to routine tissue processing procedures. After embedding in paraffin wax, tissue sections were cut at 5-μm thickness from paraffin blocks and stained with haematoxylin and eosin (H&E) (Luna, 1968). The histopathological changes in the lesioned organs were semiquantitatively scored by a trained veterinary pathologist using a brightfield microscope and their photomicrographs were taken (Olympus BX51, DP25 digital camera). The semiquantitative scoring system used was as follows: (−) none, (+) mild, (++) moderate and (+++) severe.

DNA sequencing and phylogenetic analysis
The amplified fibre-2 gene (PiAdV-A) and capsid gene (PiCV) (Freick et al., 2008) PiCV2-as AGGAGACGAAGGACACGCCTC F I G U R E 1 (a) Picture of one of the infected birds that were necropsied. Vomit from the bird is shown beside the bird's head, characterised by mucous and watery content. The bird had shrunken eyes indicating dehydration and poor condition. (b) Image of an example infected pigeon liver taken during necropsy. Widespread discoloration indicates severe necroses (white arrows) and haemorrhages on the liver distribution using the maximum-likelihood statistical method in MEGA X, respectively (Hasegawa et al., 1985;Kimura, 1980;Kumar et al., 2018).

Necropsy and histopathological findings
Macroscopically, the pigeons were dehydrated and emaciated; pectoral muscle atrophy was prominent and postmortal watery vomit was detected in two pigeons (Figure 1). The most evident gross findings were observed on livers (n = 3). Affected livers showed enlargement and fragility associated with pale, grey-yellow necrotic areas ( Figure 1) and widespread haemorrhagic spots. The proventriculus and stomach mucosae were thickened and oedematous in appearance, and in some instances, they were coated with a sticky mucous. The kidneys were enlarged and had multifocal grey foci on the serosal surfaces.

Virus isolation
The embryos were monitored every day by visualisation using an ovoscope light. Embryos started to die approximately 3 days after inoculation (for both routes, see Figure 3). After five passages in eggs, the degenerated and dead embryos' internal organs, as well as the chorioallantoic fluid, were then collected and tested by PCR to confirm the presence of different viruses; also, the chorioallantoic fluid was tested by haemagglutination assay to assess the presence of avian viruses such as Newcastle disease or avian influenza viruses. All PCR assays were negative except for the PCR test that used primers specific for PiCV. The haemagglutination assay was negative as well for any virus that could cause haemagglutination.
After inoculating the supernatants into two different cell cultures, there was no visible cytopathic effect in primary CEFCs. In contrast, supernatants caused extensive rounding, clumping and detachment of cells 48-72 h post-inoculation in the CEKC cultures (Figure 4). DNA extracted from these cultures was tested by PCR and was positive for both PiCV and PiAdV-A, suggesting the growth of both viruses in the culture together. At this point, the PiAdV-A isolate was named 'TR/SKPA20' , and the PiCV isolate was named 'TR/SKPC20' .

PCR and phylogenetic analysis of the isolates
After initially screening the supernatants generated from pigeon tissues by PCR for several different viruses, the only positive hits were for parts of the PiAdV-A fibre-2 and PiCV capsid genes ( Figure 5).
As a result of the BLAST search, the PiCV field isolate, TR/SKPC20, was found to be most closely related to strains previously isolated in Poland (MK994767, KC691682), Hungary (JF330097) and Taiwan (GO844278) (Figure 6). The PiAdV-A field isolate, TR/SKPA20, was found to be most closely related to a previously sequenced PiAdV-A strain called IDA4, with 99.03% similarity at the amino acid level (Figure 7).

DISCUSSION
In the present study, we are reporting a PiCV, PiAdV co-infection Pathological examination of the pigeons revealed severe, systemic pathological changes with a particular focus on the liver. An immunoperoxidase assay was performed on tissue sections using a serum containing antibodies that detect a common group-specific antigen of the 12 fowl adenovirus serotypes (Hess, 2000;Wan et al., 2018). The assay highlighted the presence of fowl adenovirus antigen in lesions that were visible in tissue sections from different birds that had undergone necropsy. However, because of the cross-reactivity of these antibodies, it was necessary to use molecular techniques and virus isolation to confirm precisely what virus was present (Hess, 2000).
PCR was used to screen tissue samples from the pigeons for multiple viruses and primers based on the PiAdV-A fibre-2 gene (Raue et al., 2002) and PiCV capsid gene (Freick et al., 2008), and the ampli- to isolate the suspected viruses; however, these propagation attempts were unsuccessful, suggesting that the viruses supposed to be present were either not infecting or not replicating in those cells. Although PiCV is regularly grown in culture, PiAdV serotypes are reported to be quite challenging to grow in cell culture (Vereecken et al., 1998;Duchatel et al., 2000;Marlier & Vindevogel, 2006;Schmidt et al., 2008).
The isolation of different pigeon avidenovirus strains has previously been achieved in primary CEKC and primary CELC (McFerran et al., 1976a), but the conditions used in this study were not suitable or optimal and further work is required (Raue & Hess, 1998;Takase et al., 1990;Vereecken et al., 1998). Despite this, both isolates are now available for further molecular and virological studies. Relatively little work has been carried out on either of these viruses and there is now great potential for analysis to be pursued. For example, little is understood about the fibre-2 protein of PiAdV-A, but the fibre protein of FAdV is known to play a crucial role in viral infection and pathogenesis (Lu et al., 2019;Pallister et al., 1996;Zhang et al., 2018). It is responsible for the initial attachment of the virus to cellular receptors, including the involvement of cell surface integrins (Wickham et al., 1993) and it is F I G U R E 6 Phylogenetic tree based on the amino acid sequence of the PiCV capsid gene. The tree shows circovirus species that affect fowl. The tree shows the names and GenBank accession numbers of each isolate. A red dot indicates the PiCV isolate from this study F I G U R E 7 Phylogenetic tree based on the amino acid sequence of the PiAdV fibre-2 gene. The tree shows aviadenovirus species that affect fowl. The tree shows the names and GenBank accession numbers of each isolate. The PiAdV-A isolate from this study is indicated by a red dot possible that the fibre-2 protein carries out a similar function. Considering the importance of these proteins and their involvement in infection, they may also play a role in species specificity and cross-species infection.
It is known, for example, that several FAdV serotypes 2, 4, 5, 6, 8, 10 and 12 have been previously isolated from diseased and healthy pigeons (Goryo et al., 1988;Hess et al., 1998a;Hess et al., 1998b;McFerran et al., 1976a). In this present study, other viruses such as FAdV (1 to 7, −8a and −8b, −9 to 11) and PiAdV-B and PiHV were all screened for but not detected (Table 1). In a recent study, concerning adenoviral poultry diseases, including body hepatitis (IBH), were reported for the first time in broiler and broiler breeder flocks in Turkey (Sahindokuyucu et al., 2020). PiAdV-A in pigeons is sometimes called IBH due to the intranuclear basophilic bodies seen under histopathology examination (Abadie et al., 2001;McFerran et al., 1976b), and it is possible that misdiagnosis has previously occurred and crossspecies infections are already occurring. Considering the ability of these viruses to be highly pathogenic in pigeons, it will be crucial going forward to understand the similarities between these viruses and other fowl aviadenoviruses that can infect commercially important birds, thus allowing consideration of the potential for these viruses to jump species and cause outbreaks in birds such as chickens in the future.
In a similar study, a total of 107 pigeon flocks were examined and pigeon circovirus (PiCV) genetic material was the most frequently detected, pigeon herpesvirus (PiHV) genetic material was second in frequency, while genetic material of pigeon aviadenovirus was found only in two flocks of young birds with clinical symptoms of YPDS. Moreover, the presence of fowl aviadenovirus (FAdV) genetic material was not detected in any of the studied flocks (Stenzel et al., 2012). Our study reports the first identification and isolation of PiAdV-A and PiCV in a pigeon flock in Turkey to the best of our knowledge. In contrary to previous studies, PiHV and FAdV genetic material were not detected in the tested flocks.

CONCLUSION
The clinical signs observed in pigeons of this study would typically be associated with Young Pigeon Disease Syndrome (YPDS), a multifactorial disease in which PiCV has been shown to play an important role. PiCV is associated with immunosuppression, making the birds more susceptible to secondary bacterial, viral or parasitic infections.
This study indicates that co-infections of PiAdV and PiCV are possible and are associated with a severe, sometimes fatal disease in the birds infected. Potentially, an initial PiCV infection weakened the flock, making it more susceptible to a PiAdV infection, and this enhanced the clinical disease that occurred in the birds. While merely speculation, it suggests a possible mechanism that would explain this devastating multipathogen infection in the flock.
It is crucial in the future to measure the prevalence of both viruses in pigeon populations to determine their pathogenic burden. Experimental co-infections to tease apart the pathogenesis of disease will be essential so that if this happens more frequently or starts to create problems in commercially important fowl, then it can be recognised quickly and tackled effectively. This study paves the way for these future studies and offers the potential for a better understanding of viral infections of birds in Turkey.