Cardiac Lesions of Natural and Experimental Infection by Parrot Bornaviruses

J. Comp. Path. 2020, Vol. 174, 104e112

Available online at www.sciencedirect.com

ScienceDirect www.elsevier.com/locate/jcpa

INFECTIOUS DISEASE

Cardiac Lesions of Natural and Experimental Infection by Parrot Bornaviruses J. Leal de Araujo*, S. S. Hameed†, I. Tizard‡, P. Escandon‡, P. R. Giaretta‡, J. J. Heatleyx, S. Hoppes‡ and R. R. Rech‡ *Departamento de Ci^encias Veterinarias, Universidade Federal da Paraiba, Areia, Paraiba, Brazil, † College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq, ‡ Department of Veterinary Pathobiology and x Department of Small Animal Clinical Sciences, Texas A&M University, College Station, Texas, USA

Summary Lymphoplasmacytic inflammation associated with bornavirus N protein occurs in the epicardial ganglia, myocardium and endocardium of birds diagnosed with proventricular dilatation disease (PDD). These pathological findings suggest that sudden death in psittacine birds might stem from cardiac compromise due to parrot bornavirus (PaBV) infection. Therefore, we investigated cardiac lesions in cases of PDD, searching databases from 1988 to 2019, and reviewed three experimental studies of PaBV infection. Fifty cases of PDD in birds infected naturally with PaBV and 27 cases of PDD in birds infected experimentally with PaBV (all having descriptions of inflammatory cardiac lesions) were reviewed. For each case, five regions of the heart were evaluated by light microscopy and immunohistochemistry (IHC). These regions were the epicardial ganglia/nerves, the endocardium, the myocardium, the Purkinje fibres and the great vessels. Sudden death was documented in 17/50 naturally infected cases, while 23/50 had digestive signs, and only 12/50 had neurological signs. Grossly, only five naturally-infected and five experimentally-infected cases had cardiomegaly or hydropericardium. Epicardial ganglioneuritis was the most consistent microscopical finding in natural (46/50) and experimental cases (26/27), followed by myocarditis (34/50) for naturally-infected and endocarditis for experimentally-infected birds (6/27). PaBV-2 antigen was detected most frequently by IHC in the epicardial ganglia (54/77) compared with the other tissues. This retrospective study demonstrates the presence of PaBV protein and inflammation in the heart of birds infected with PaBV and suggests a link between PaBV and cardiac disease and sudden death in psittacine birds. Ó 2019 Elsevier Ltd. All rights reserved. Keywords: epicardial ganglioneuritis; myocarditis; proventricular dilatation disease; sudden death

Introduction Parrot bornaviruses (PaBVs) belong to the species Pisttaciform 1 and 2 of the recently classified genus Orthobornavirus, which encompasses mammalian and avian bornaviruses (Amarasinghe et al., 2018). In birds, PaBV causes a lethal disease commonly referred to as proventricular dilatation disease (PDD) (Tizard et al., 2016; Amarasinghe et al., 2018). This disease affects captive populations of Correspondence to: R. R. Rech (e-mail: [email protected]). 0021-9975/$ - see front matter https://doi.org/10.1016/j.jcpa.2019.11.008

psittacine birds worldwide and is characterized by neurological and/or gastrointestinal signs (Hoppes et al., 2010). Gross lesions are often limited to enlargement of the proventriculus and/or crop of affected birds and emaciation (Hoppes et al., 2010; Tizard et al., 2016). The most common microscopical lesions are lymphoplasmacytic infiltration of the central nervous system (CNS) and ganglia of gastrointestinal (GI) organs such as the crop, proventriculus, ventriculus and intestines. However, inflammatory lesions in the ganglia and nerves of the adrenal gland, eyes and heart have also been Ó 2019 Elsevier Ltd. All rights reserved.

Cardiac Lesions in Parrot Bornavirus Infection

described (Ouyang et al., 2009; Raghav et al., 2010; Wunschmann et al., 2011; Payne et al., 2011b). Avian autonomic cardiac innervation consists of sympathetic nerves arising from the sympathetic chain of the spinal cord; and parasympathetic nerves arising from the vagus nerve (Dzialowski and Crossley, 2015). After experimental intramuscular inoculation in cockatiels (Nymphicus hollandicus), PaBV has been demonstrated to travel first to the CNS and then to peripheral organs such as the GI tract and periadrenal ganglia. Occurrence of PaBV in the vagus nerve has been detected as early as 35 days post inoculation (dpi), followed by the spread to the heart at 40 dpi (Leal de Araujo et al., 2017b). Several studies have reported cardiac lesions in PaBV-infected psittacine birds and viral antigen has been detected in the epicardial ganglia, the myocardium and endocardium, which may be accompanied of lymphoplasmacytic infiltrates (Ouyang et al., 2009; Raghav et al., 2010). Additionally, sudden death has also been reported in birds with PDD (Lierz, 2015). Therefore, cardiac dysfunction in PDD could play a role in the declining health of infected birds; however, it is not clear how often the heart is affected in PDD. The aim of this study was to evaluate cardiac lesions in PDD and to determine whether PaBV nucleoprotein is associated with these lesions in both naturally-infected and experimentally-infected birds. We also reviewed the clinical signs shown by these birds. We hypothesized that PaBV antigen detected by immunohistochemistry (IHC) and lymphoplasmacytic infiltrates would be present in both natural and experimentally-induced PDD and that sudden death would be a common clinical sign associated with the presence of cardiac lesions.

Materials and Methods Case Material

Natural cases were accessed from the avian pathology archives of the Schubot Exotic Avian Health Center (1988e1998) and the necropsy records from the Department of Veterinary Pathobiology (1999e2018), College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, Texas, USA. Search terms included ‘PDD’, ‘proventricular dilatation disease’, ‘macaw wasting disease’ and ‘avian borna virus’. Naturally occurring cases of PDD were evaluated and filtered for histological descriptions of lymphoplasmacytic infiltrates in the heart. For inclusion in the study, cases must have had evidence of classic lesions of PDD in the GI tract. From these cases, histological slides of the heart were re-evaluated to confirm the presence of inflammatory lesions. Fifty cases of PDD with descriptions of cardiac

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lesions were selected for additional evaluation based on histological and immunohistochemical evaluation. Necropsy accession histories were reviewed with attention to species, age, sex and for client-reported or clinician-reported clinical signs related to GI or neurological dysfunction or sudden, acute or unexpected death. We equated the following terms with sudden death: ‘sudden death’, ‘acute death’, ‘found dead’, ‘death on examination’, ‘died shortly after presentation’, ‘sudden collapse’ or ‘died on the way to the clinic’. We equated the following terms with wasting (W): ‘wasting away’, ‘weight loss’, ‘progressive weight loss, emaciation/cachexia’, which was included as a GI sign. For neurological signs we included: ‘ataxia’, ‘falling off the perch’, ‘visual impairment’, ‘seizures’, ‘lethargy or depression’ and ‘weakness’. Cockatiels used in three previous experimental studies were also evaluated retrospectively for cardiac lesions (Leal de Araujo et al., 2017b; Hameed et al., 2018). These birds had been inoculated intramuscularly with PaBV-2. Hearts of 27 cockatiels with descriptions of cardiac lesions were re-evaluated for gross and microscopical cardiac lesions, and the presence of PaBV by IHC. Histopathology and Immunohistochemistry

Serial transverse sections, from the great vessels to apex, were obtained from the hearts (Fig. 1). Five cardiac structures were evaluated by light microscopy: the epicardial ganglia/nerve, the endocardium, the myocardium, the Purkinje fibres and the great vessels. All sections evaluated for inflammatory lesions were also tested for PaBV N-protein by IHC. Briefly, IHC was performed using polyclonal antibodies against a specific region of the PaBV N-protein on serial tissue sections, mounted on charged slides (Leal de Araujo et al., 2017b; Hameed et al., 2018). Due to the intranuclear replication site of PaBV, only cells with intranuclear or intranuclear and intracytoplasmic immunolabelling were considered positive, while cells with only intracytoplasmic immunolabelling were considered negative (Raghav et al., 2010). For negative controls, a tissue section from a cockatiel known to be positive for PaBV without the primary antibody, and a tissue section from a cockatoo (Cacatua moluccensis) known to be negative for PaBV and treated with the primary antibody were used. Reverse Transcriptase Polymerase Chain Reaction

Cardiac tissues from birds in experimental studies one and three were analysed retrospectively for by reverse transcriptase polymerase chain reaction (RT-PCR) targeting the PaBV matrix (M) protein gene and

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the phosphoprotein (P) gene (Guo et al., 2014; Hameed et al., 2018). We also investigated PCR results from study 2, where cardiac tissues were tested in duplicate for expression of the genes encoding the M and the P proteins. Briefly, mRNA encoding the PaBV P protein was detected by using a Taqman RT-PCR assay performed with TaqManÒ Fast Virus 1-Step Master Mix (Invitrogen, Carlsbad, California, USA) and PaBV P primers: 50 AAGAAGAA[Y]CC[Y]TCCATGATCTC-3 and 50 -AA[Y]TGCCGAAT[B]A[R] GTCATC-30 and a Taqman probe (50 -FAM-TCGATAACTG [Y]TCCCTTCCGGTC-BHQ-30 ). mRNA encoding the PaBV M protein was detected by using PaBV M primers: 50 -

GGTAATTGTTCCTGGATGG-30 and 50 -ACACCAATGTTCCGAAGACG-30 , and a Taqman probe (50 -FAM-TCGATAACTG [Y] TCCCTTCCGGTC-BHQ-30 ).

Results Clinical Data

The clinical history of the 50 naturally-occurring cases (Table 1) showed that 31 birds had signs of GI tract dysfunction, including emaciation, anorexia, regurgitation, diarrhoea or undigested seeds in the faeces. Only 18 birds had neurological signs, which included ataxia, lethargy, depression and seizures, and 13 birds had both GI and neurological signs.

Fig. 1. Sampling technique of the heart for this study. (A) Serial sections were collected from the great vessels of the heart to the apex, beginning with a transverse section to include both ventricles (B). This transverse section (C) facilitates the visualization of multiple epicardial ganglia (inset) on histological evaluation, especially in the hearts of the larger species (macaws, cockatoos, African grey parrots).

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Cardiac Lesions in Parrot Bornavirus Infection Table 1 Common clinical signs of psittacine species with naturally occurring proventricular dilatation disease Bird 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

Genus

Sex

Age

Sudden death

GI signs

Neurological signs

Cockatoo (Cacatua sp.) Cockatoo (Cacatua sp.) Cockatoo (Cacatua sp.) Cockatoo (Cacatua sp.) Cockatoo (Cacatua sp.) Cockatoo (Cacatua sp.) Cockatoo (Cacatua sp.) Cockatoo (Cacatua sp.) Cockatoo (Cacatua sp.) Cockatoo (Cacatua sp.) Cockatoo (Cacatua sp.) Macaw (Ara sp.) Macaw (Ara sp.) Macaw (Ara sp.) Macaw (Ara sp.) Macaw (Ara sp.) Macaw (Ara sp.) Macaw (Ara sp.) Macaw (Ara sp.) Macaw (Ara sp.) Macaw (Ara sp.) Macaw (Ara sp.) Macaw (Ara sp.) Macaw (Ara sp.) Macaw (Ara sp.) Macaw (Ara sp.) Psittacus sp. Psittacus sp. Psittacus sp. Psittacus sp. Psittacus sp. Psittacus sp. Psittacus sp. Psittacus sp. Psittacus sp. Psittacus sp. Psittacus sp. Psittacus sp. Psittacus sp. Psittacus sp. Alisterus sp. Aratinga sp. Aratinga sp. Guarouba sp. Guarouba sp. Thectocercus sp. Myopsitta sp. Myopsitta sp. Nymphycus sp. Poicephalus sp.

F F F NI F NI M NI NI F F M NI F M F M M F F NI M NI NI NI NI F F NI F F F M M M F M M M M F M F M F M NI F F NI

8 years 10 years NI 8 weeks NI 1.5 years 30 years 7 months 5 weeks 17 years 4 months 5 months 1 year 20 weeks 15 years 7 months 4 years NI NI 8.5 years 8 months 7 months 8 years 21 years 2.5 years 38 years 5 months 4 years <2 years 5 years NI NI NI 2 years 5 months NI 6 months 12 years NI NI 1 years <1 year NI 4 years 1.5 years 2 years 4 weeks 15 years 5 years NI

e e SD e SD e e e SD e e e SD e e e e SD SD e e SD e SD e SD e e SD e e e SD SD e SD e e e e SD e SD SD e e SD e e e

W W e R R W A, W W, D, R R R WA, R R, W e A, CSD V,D R W, A e e A, USF W,D,R R R, CSD e e e R, W W A W e A, W, USF e W W e R e e e e W e e W, USF A, R e e W e

LD WE e WE e e e e e e LD e e e L LD, S e e e LD e e LD e e FDB L AT WE e S WE e e e e L AT, VI, FDB FDB e e e e e e e e L e e

F, female; M, male; NI, no information; SD, sudden death; GI, gastrointestinal; W, wasting; R, regurgitation, AN, anorexia; D, diarrhoea; CSD, crop stasis or distension; V, vomiting; USF, undigested seeds in faeces; WE, weakness; AT, ataxia/falling off perch; VI, visual impairment; LD, lethargy or depression; FDB, feather-damaging behaviour; S, seizures.

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Sudden death was described in 17 cases (34%), while feather damaging behaviour was reported in three cases. An echocardiogram of a naturally-occurring case of PDD revealed evidence of tachycardia (i.e. heart rate of 375 beats/min post sedation) with occasional arrhythmia, pericardial effusion, ventricular dilation, decreased ventricular fractional shortening (20%) and mitral regurgitation, suggestive of cardiac insufficiency. The African grey parrot (Psittacus erithacus) was the most frequent species (14 individuals) and also the species to most often present with sudden death (five cases), followed by the blue and gold macaw (Ara ararauna) (five individuals and one case of sudden death). In experimental study 1, one cockatiel presented with acute signs of depression and lethargy and died shortly thereafter. Another was found dead without any prior signs of illness. In study 3, all eight cockatiels presented with classical GI signs of PDD, including weight loss and undigested seeds in the faeces. Out of the 50 birds, 21 were female and 16 were male; however, sex was not recorded for 13 cases. Gross Lesions

Gross cardiac lesions were found in five naturallyinfected cases. Cardiomegaly was observed in three cases, including a PaBV-positive Quaker parrot (Myopsita monachus) that was presented with an enlarged right atrium (Fig. 2A), and an African grey parrot with cardiomegaly. Hydropericardium was reported in two cases. In the experimentally-infected cases, cardiomegaly (Fig. 2B) was seen in four birds and hy-

dropericardium (Fig. 2C) was documented in one case.

Histopathology and Immunohistochemistry

Microscopical lesions and PaBV immunolabelling showed that 95% of the naturally-infected cases of PDD with cardiac lesions had lymphoplasmacytic infiltration of the ganglia (see Fig. 3A) and nerves of the epicardium (Table 2). Myocarditis (Fig. 3C) was present in 68% of these birds, while Purkinje fibres were effaced by lymphoplasmacytic infiltrates in only 36% of the cases. Mural endocarditis was observed in 20% of all cases of PPD with cardiac lesions. Eight cases had ‘pan-carditis’. Additionally, 18% of the cases had lymphoplasmacytic infiltration of the tunica media of the great cardiac vessels. No inflammatory lesions were observed in the valves. Of the 46 cases of epicardial ganglioneuritis, 30 were also positive for PaBV-2 IHC (Fig. 3B), while of the 34 cases with myocarditis, 20 had positive immunolabelling (Fig. 3D). For the endocardium and Purkinje fibres, only 20% and 23%, respectively, of the cases with inflammation in these areas were also positive for PaBV-2. For the tunica media of the great vessels, although only nine cases had perivascular lymphoplasmacytic infiltrates, PaBV-2 antigen was detected in 15 cases. All naturallyinfected African grey parrots had ganglioneuritis in the epicardium and eight of the 14 African grey parrots also had myocarditis. Four of five blue and gold macaws had ganglioneuritis in the epicardium or myocarditis.

Fig. 2. Gross cardiac lesions in psittacine birds with PDD were uncommon. Heart sections from three different cases are shown. An enlarged right atrium was apparent in a natural (A; photograph courtesy of Dr. L. Bryan) and an experimental case (B), and hydropericardium was documented in one experimental case (C).

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Cardiac Lesions in Parrot Bornavirus Infection

Fig. 3. Histological inflammatory lesions in the heart of psittacine birds with PDD. (A) Epicardial ganglioneuritis was the most frequent histological finding and the cellular infiltrate was comprised mostly of lymphocytes and plasma cells. HE. Bar, 20 mm. (B) PaBV-2 antigen in the nucleus and cytoplasm of neurons and inflammatory cells. IHC. Bar, 20 mm. (C) Lymphoplasmacytic myocarditis. HE. Bar, 50 mm. (D) Inflammatory cells and cardiomyocytes immunolabelled for PaBV-2. IHC. Bar, 50 mm.

In experimental study 1, 12 cockatiels had lymphoplasmacytic infiltration of the heart, but had lymphoplasmacytic infiltration of the nerves and/or epicardial ganglia. Lymphoplasmacytic infiltrates in the myocardium and endocardium were present in 34% of the cockatiels. No inflammatory lesions were observed in the Purkinje fibres or great vessels. In experimental study 2, seven cockatiels had lymphoplasmacytic infiltration of at least one of the five regions evaluated. Six cockatiels (85.7%) had lym-

phoplasmacytic inflammation of the ganglia and/or nerves of the epicardium. This type of inflammation also occurred in the myocardium and Purkinje fibres of two cockatiels. No inflammatory lesions were present in the myocardium or endocardium of the cockatiels from this study. In experimental study 3, all eight cockatiels developed lymphoplasmacytic infiltration of the heart with further such inflammation in the ganglia and/or nerves of the epicardium. These infiltrates were also

Table 2 Psittacine birds with inflammatory lesions and positive bornavirus antigen immunolabelling in the heart Epicardial ganglia Natural cases (n ¼ 50) HE 46 IHC 30 Experimental study I (n ¼ 12) HE 12 IHC 10 Experimental study II (n ¼ 7) HE 6 IHC 6 Experimental study III (n ¼ 8) HE 8 IHC 8

Myocardium

Endocardium

Purkinje fibres

Great vessels

34 20

10 2

17 4

9 15

4 4

4 1

0 0

0 2

0 0

1 1

1 1

0 0

1 1

1 1

0 0

0 0

J. Leal de Araujo et al.

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observed in the myocardium of one cockatiel and in the endocardium of another. No inflammatory lesions were observed in the Purkinje fibres or great vessels. For the experimental studies, only the first study had differences between the number of birds with inflammatory lesions and the number of birds with positive IHC. Ten birds were positive for PaBV immunolabelling of the epicardial ganglia, while four cases were positive for the myocardium, one case was positive for the endocardium and two cases were positive for the great vessels. No immunolabelling was detected in the Purkinje fibres. In the other two experimental studies, immunolabelling was present in all regions with inflammation. Reverse Transcriptase Polymerase Chain Reaction

All 27 samples from the experimental studies tested by RT-PCR were positive for expression of PaBV genes.

Discussion We retrospectively analysed cardiac lesions in 77 psittacine birds that were infected with PaBV and/or had a diagnosis of PDD, including both natural and experimental infections, over a period of 31 years from two different necropsy databases and tissue banks. Out of 50 naturally-infected cases, 34 dated from before PaBV was recognized as the causative agent of PDD (Honkavuori et al., 2008; Kistler et al., 2008). In these cases, IHC was used to confirm PaBV as the cause of ‘macaw wasting syndrome’. We identified lymphoplasmacytic infiltration of the epicardium, myocardium, endocardium, Purkinje fibres and/or cardiac great vessels. These results associate inflammatory lesions of the heart in psittacine birds with PDD, as described elsewhere (Raghav et al., 2010; Heffels-Redmann et al., 2011; Payne et al., 2011a; Leal de Araujo et al., 2017a). In the 27 experimentally-infected cases, epicardial ganglioneuritis was the most consistent finding, representing 97% of the cardiac lesions. Some of the experimentally-infected cockatiels lived as few as 114 dpi, which might not have been sufficient time for PaBV to spread to other regions of the heart. There is individual and interspecific variation, but generally, epicardial ganglia in avian species are concentrated in the dorsal areas, over the posterior atrial walls and near the great vessels of the heart (Smith, 1971). Cardiac diseases in pet and wild birds may be due to infectious, congenital, toxic, neoplastic or idiopathic causes. Two studies have systematically searched for cardiac lesions in psittacine birds submitted for necropsy examination. In one study, 26

(9.7%) of 269 psittacines subjected to necropsy examination had evidence of cardiac disease (Oglesbee and Oglesbee, 1998). In another study of 107 psittacines submitted for necropsy examination, 36% had gross lesions in the heart, great vessels of the heart, or both (Krautwald-Junghanns et al., 2004). The main histological findings reported for these birds included myocarditis (59.8%), focal coagulative necrosis (23.4%) and lipomatosis cordis (48.6%). However, these studies were published before PaBV was known to be the causative agent of PDD. We found a reasonable incidence of myocarditis; >10%, or seven of 50 cases of naturally-occurring PDD. We suggest that the lack of myocarditis in experimentally-infected cases may have been due to the relatively short experimental time (generally <200 days), innate natural resistance of the species used and the bornavirus genotype or entry route of naturally-infected cases, which remains relatively uninvestigated. Although not frequently reported in pet birds, cardiomegaly was observed in our study. Hypertrophic and dilated cardiomyopathy are described uncommonly in parrots (Schmidt et al., 2015). In Mynah birds (Acridotheres spp.), cardiomegaly is reported more commonly and is associated with haemochromatosis (Ensley et al., 1979; Rosenthal and Stamoulis, 1993). Dilated cardiomyopathy in young turkeys, also known as ‘round heart disease’, frequently causes cardiomegaly and sudden death (Beaufrere and Wakamatsu, 2014). Although described in chickens (Gallus domesticus), round heart disease is considered rare in this species (Julian, 2005). In one study evaluating congestive heart failure in African grey parrots, identification of the aetiology of cardiac disease was not performed; however, one of the parrots suffered from advanced PDD, with cardiomegaly and lymphoplasmacytic infiltrates in the myocardium and epicardial ganglia found on post-mortem examination (Juan-Salles et al., 2011). The number of African grey parrots positive for PaBV with cardiac lesions in this study suggests that some psittacine species may be more likely to develop inflammatory cardiac lesions than others. However, our study numbers were small, with similar percentages of macaws (5/13) and cockatoos (3/11) presenting with sudden death. PaBV-2 antigen was not detected by IHC in the heart of all naturally-infected cases with inflammatory lesions. There are two possible explanations for this observation, either or both of which may have contributed to this finding. Firstly, some of the formalin-fixed and paraffin wax-embedded (FFPE) tissues used in this study were collected over 30 years ago, which might have interfered with or otherwise reduced the immunoreactivity of the sections via a phenomenon known as antigen decay (Grillo et al., 2015). Secondly, other PaBV

Cardiac Lesions in Parrot Bornavirus Infection

genotypes (PaBV genetic diversity) could have been involved in some of these cases, leading to lack of cross-reaction with the antibody used in the study. Experimentally, different PaBV genotypes likely associate with different clinical manifestations (Piepenbring et al., 2016), with clinical signs of either a digestive or neurological nature. Some genotypes of PaBV could have a more pronounced cardiac tropism, but more study is needed to determine this. Interestingly, PaBV-2 antigen was detected in the tunica media of great vessels in six naturallyinfected cases and two experimentally-infected cases, which lacked perivascular cuffing or vasculitis. It might be suggested that the lack of PaBV immunoreactivity in some cardiac inflammatory lesions supports the hypothesis that another infectious agent is responsible for PaBV/PDD; however, all of the present cases were diagnosed as PDD positive via histopathology. For future studies we recommend that genotype determination is included so that clinical, gross and histopathogical changes and genotype associations may be investigated further. In cell culture, PaBV is non-cytopathic and the behaviour of the virus in vivo appears similar (Tizard et al., 2016; Murray et al., 2017). Injury to the tissues is likely caused by T-cell infiltration, rather than a virus-induced lesion (Hameed et al., 2018). As seen in the present study, PaBV can be detected in tissues without associated inflammatory or circulatory lesions. More immunopathological studies are required to clarify the correlation between PaBV infection and gross, histopathological and clinical manifestations of disease. This retrospective study supports the hypothesis that PaBV may be involved in the development of cardiac disease in psittacine birds, and that PDD must be included as a differential diagnosis in cases of sudden death or when the necropsy examination reveals cardiac alterations such as cardiomegaly or hydropericardium. Because epicardial ganglioneuritis can be considered a diagnostic histological lesion of PDD in both natural and experimental cases, serial sections of the heart between the great vessels and the level of the atrioventricular valves are recommended for routine post-mortem screening of birds affected by sudden death or those with clinical signs suggested of PDD.

Acknowledgments The authors thank D. Turner and the staff of the Veterinary Pathobiology Histology Laboratory for technical support throughout this study, and the Schubot Center for Avian Heath for funding this project.

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July 9th, 2019 ½ Received, Accepted, November 26th, 2019