Diagnosis of Adenovirus Infections in Psittacine Birds by DNA in situ Hybridization

Kenneth S. Latimer, Frank D. Niagro, Raymond P. Campagnoli, and Antonio Ramis

Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7388 USA (Latimer, Campagnoli); Department of Clinical Investigation, Eisenhower Army Medical Center, Building 332, Fort Gordon, GA 30905-5650 USA (Niagro); Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain (Ramis).

Abstract. Aviadenovirus infection was diagnosed in nine psittaciform birds using DNA in situ hybridization. Two DNA probes, designated FN-23 and FN-96 were used for this study. Neither of the DNA probes showed cross hybridization polyomavirus or psittacine beak and feather disease viral nucleic acids. Probe FN-23 did cross hybridize weakly to herpesvirus nucleic acid from 2 of 8 psittaciform birds with Pacheco's parrot disease. The results of this study indicate that probe FN-96 is a more satisfactory diagnostic reagent to detect adenovirus infection in psittaciform birds.

Introduction

Aviadenoviruses are icosahedral, non-enveloped, 74 to 80 nm diameter particles that contain a double-stranded, linear, 30-37 kilobase pair DNA genome. ^1,2 These viruses have a worldwide distribution and are responsible for latent or clinically severe infections, depending upon viral tropism. ² Naturally-acquired, aviadenovirus infections in poultry may be associated with hepatitis, splenitis, enteritis, ventriculitis, pancreatitis, or altered reproduction ("egg drop syndrome"). ^2-4 Adenovirus infections in psittaciform birds may be an incidental finding or may be associated with inclusion body pancreatitis, encephalitis, hepatitis, splenitis, hemorrhagic enteritis, inclusion body nephrosis, or conjunctivitis. ^5-8

Aviadenovirus infections usually are diagnosed by virus isolation and serotyping, agar gel immunodiffusion assay, hemagglutination antibody titers, or ELISA testing using fresh tissues and sera. ^2,3,9,10 Diagnostic pathologists often receive only formalin-fixed tissues for analysis. Therefore, the disease diagnosis depends upon routine histopathology or electron microscopy. Histologically, adenoviral inclusions in H&E-stained tissue sections may exhibit variable size and in tinctorial properties, ranging from enlarged and basophilic to small and eosinophilic. ^11,12 Because of their variable appearance, aviadenovirus inclusions may be mistaken for those of herpesvirus, circovirus, or polyomavirus in psittaciform birds. Transmission electron microscopy, using ultrathin tissue sections or negatively stained tissue extracts, is expensive and not universally available; however, virion location, size, capsid morphology, and arrangement are fairly characteristic for aviadenoviruses. ^2,3

Recent histologic advances in the diagnosis of aviadenovirus infection include immunoperoxidase staining ^4,9,13,14 and immunofluorescence techniques. ^2,15,16 Major disadvantages of immunoperoxidase staining include lack of monoclonal antibody recognition of a given epitope, excessive nonspecific (background) staining, loss of antigenicity following formalin fixation, inability to detect latent viral infection, and disposal of carcinogens such as diaminobenzidine chromagen solutions. Immunofluorescence techniques usually require frozen, unfixed tissue; may suffer from excessive nonspecific staining; and the fluorochrome marker may fade with prolonged or repeated exposure to ultraviolet light. ¹⁷ The following study uses by DNA in situ hybridization with digoxigenin-labeled oligonucleotide probes to diagnose adenoviral infections in formalin-fixed, paraffin-embedded tissues from various genera and species of psittacine birds.

Materials and Methods

Tissues and study population: Formalin-fixed, paraffin embedded tissue specimens from birds with confirmed or suspected adenovirus infections were obtained from archival storage at the Department of Pathology, College of Veterinary Medicine, University of Georgia. All tissue specimens had been obtained from psittacine birds with naturally-acquired disease. The study population consisted of tissues from 9 birds: 2 Lilac-crowned Amazon Parrots (Amazona finschi), 1 African Grey Parrot (Psittacus erithacus erithacus), 1 Cockatiel (Nymphicus hollandicus), 1 Cockatoo (Cacatua sp. ), 1 Jardine's Parrot (Poicephalus gulielmi), 1 Lovebird (Agapornis sp. ), 1 Red-bellied Parrot, (Poicephalus rufiventris), and 1 Senegal Parrot (Poicephalus senegalus). Tissues from chickens (Gallus gallus domesticus), bobwhite quails (Colinus virginianus), and a pigeon (Columba livia) with confirmed aviadenovirus infection served as positive control specimens. Negative control specimens consisted of tissues from naïve birds.

Archived paraffin embedded tissues also were obtained from birds with Pacheco's parrot disease virus (herpesvirus, n = 8), polyomavirus (n = 3), and psittacine beak and feather disease virus (n = 1) to determine if either of the DNA probes would cross hybridize with these common pathogens. This study population included 12 birds: 2 Double Yellow-headed Amazon Parrots (Amazona ochrocephala oratrix), 1 African Grey Parrot, 1 Timneh African Grey Parrot (Psittacus erithacus timneh), 1 Amazon Parrot (Amazona sp. ), 1 Cockatiel (Nymphicus hollandicus), 2 Cockatoos (Cacatua sp. ), 1 Moluccan Cockatoo (Cacatua moluccensis), 1 Sulphur-crested Cockatoo (Cacatua galerita), 1 Macaw (Ara sp. ), and 1 Rose-ringed parakeet (Psittacula krameri manillensis).

Routine histopathology: Paraffin-embedded tissue blocks were sectioned at 3 µm and stained with hematoxylin and eosin. Tissues initially were reviewed to confirm the presence of viral inclusions. Subsequently, replicate tissue sections were cut and placed on ProbeOn Plus^a glass microscope slides for DNA in situ hybridization.

DNA probes: The two DNA probes designed for use in this study had the following designations and nucleic acid sequences:

Nucleotide sequence data for psittacine adenoviruses was not found in the GenBank archives. Therefore, probes FN-23 (36 bases) and FN-96 (40 bases) were designed from published sequence data for the chicken adenovirus (type 10) penton gene (GenBank accession M87008). ¹⁷ Both oligonucleotide DNA probes were based upon unique penton gene nucleotide sequences which code for a structural capsid protein. Probe FN-23 was located near the center of the penton gene, while probe FN-96 was located toward the end of the penton gene (which codes for the carboxyl end of the structural protein). Both probes were designed to provide maximum cross-reactivity to detection of adenovirus infections in various genera and species of psittacine birds. Cross-reactivity of the probe would depend upon complementary nucleotide sequences coding for the penton protein. Prior to use, the probes were labeled at the 3' end with digoxigenin using a commercially available kit. ^c

DNA in situ hybridization: A MicroProbe work station^a was used to facilitate handling of tissue sections, to control reaction temperatures, and to minimize reagent use. Tissue sections were deparaffinized in a 3:1 solution of limonene:xylene and rehydrated by passage through graded alcohols to Automation buffer. ^b To enhance probe penetration, the tissue sections were digested in 0.3% pepsin in Automation buffer (pH 2.0) for 10 minutes at 37°C. Prehybridization was performed using 100% formamide treatment for 10 minutes at 105°C. The tissue sections subsequently were hybridized to the digoxigenin-labeled oligonucleotide probes for 10 minutes at 105°C and then for 45 minutes at 37°C. The probes were at a 10 to 20 nM concentration in a solution of 22.5% deionized formamide, 7.5% chondroitin sulfate, 5x SSC (standard sodium citrate), and 50 mM sodium phosphate. Following stringency washes of 0.5X and 0.2X SSC at 37°C, antidigoxigenin antibody conjugated to alkaline phosphatase was applied to the tissue sections. Nitroblue tetrazolium dye served as the chromagen. Foci of dye reduction to blue-black formazan pigment corresponded to the presence of adenoviral nucleic acid. Tissue sections subsequently were counterstained with fast green, dehydrated through graded alcohols to xylene, coverslipped, and examined with a light microscope.

Results

The data on DNA probe reactivity are presented in Tables 1 and 2. The pattern and staining intensity of the probes varied from finely stippled and light to diffuse and dark. The positive control tissues had dark diffuse formazan staining, while all negative control tissues were devoid of formazan deposition.

The H&E-stained tissues from all birds with adenovirus infection (Table 1) had intranuclear inclusions that varied in size and tinctorial property. Some intranuclear inclusions filled the nucleus and marginated the chromatin, but were unaccompanied by karyomegaly. In most birds, however, adenovirus infection was associated with marked karyomegaly (Figs. 1 & 2). The color of the nuclear inclusions ranged from eosinophilic to basophilic; larger inclusions were more basophilic. Following DNA in situ hybridization, the presence of aviadenovirus nucleic acid was confirmed by the deposition of blue-black formazan pigment (Figs. 3 & 4).

Fig. 1. African Grey Parrot, liver, adenovirus infection, H&E stain. Multifocal hepatocellular necrosis with two karyomegalic nuclear inclusions (center).	Fig. 2. African Grey Parrot, liver, adenovirus infection, probe FN-96, antidigoxigenin antibody-alkaline phosphatase indicator system, NBT chromagen, and fast green counterstain, Chromagen deposition identifies adenoviral DNA within hepatocyte nuclear inclusions.

Fig. 3. Senegal Parrot, small intestine, adenovirus infection, H&E stain. Karyomegalic adenoviral nuclear inclusions are present within some enterocytes.	Fig. 4. Senegal Parrot, intestine, probe FN-96, antidigoxigenin antibody-alkaline phosphatase indicator system, NBT dye chromagen, and fast green counterstain. Chromagen deposition confirms adenoviral DNA within enterocyte nuclear inclusions.

Probe FN-96 resulted in the best formazan deposition in psittaciform tissues. Probe FN-23 detected fewer birds with adenovirus infection and displayed weaker hybridization and chromagen deposition. Probe FN-96 did not hybridize to tissues from psittaciform birds with polyomavirus infection (n=3) or Pacheco’s parrot disease (n=8). Probe FN-23 also failed to hybridize with tissues of polyomavirus-infected birds, but did cross-hybridize to tissue specimens from 2 of 8 psittaciform birds with Pacheco's parrot disease (herpesvirus infection).

Discussion

Randomly primed DNA probes generated from purified viral DNA have been used experimentally diagnose adenovirus infections in fowl. ¹⁸ Results of that study indicated that DNA probes were more sensitive than immunoperoxidase staining in detecting aviadenovirus infections. In addition, Allan et al also found that nonradiolabeled probes were more sensitive than ³⁵S-labeled probes in diagnosing adenovirus infection and required a shorter turnaround time. ¹⁸

The present study demonstrates that synthesized oligonucleotide probes also can successfully detect avian adenovirus infections. In addition, the design of these probes allows cross-detection of adenovirus infections in various genera and species of psittacine birds. The reactivity of probes FN-23 and FN-96 (designed from chicken nucleotide sequences but applied to psittacine tissues), reflects homologous nucleotide sequences within the penton base coding region of the aviadenovirus genome.

In psittaciform birds, probe FN-96 may have diagnostic use in confirming adenovirus infection, even in the presence of concurrent polyomavirus or herpesvirus (Pacheco's disease) infections. In contrast, probe FN-23 may be marginally useful in diagnosing adenovirus infection because of cross detection of herpesvirus nucleic acid. Although cross reactivity was only observed in tissues from two of eight psittaciform birds, this may be explained by detection of psittacine herpesvirus subtypes ¹⁹ or by the presence of concurrent asymptomatic adenovirus infections in these birds. The possibility of concurrent adenovirus infection, however, is not supported by the hybridization results of probe FN-96. Despite cross reactivity, probe FN-23 has been used successfully to detect hepatic, renal, and intestinal adenovirus infections in eclectus parrots (Eclectus roratus). In that instance herpesvirus nucleic acid could not be detected by application of another herpesvirus-specific DNA probe (FN-49) to replicate tissue sections. ²⁰

The major attributes of DNA in situ hybridization in a diagnostic laboratory setting include definitive diagnosis of specific viral infections from formalin-fixed tissues, good sensitivity (detection of 10 to 30 copies of viral DNA per focus of infection),²¹ and the ability to accomplish retrospective disease studies. In summary, DNA in situ hybridization is a relatively simple technique that may be incorporated into the diagnostic functions of any histopathology laboratory with only modest expenditure for additional reagents, supplies, and equipment.

Sources and manufacturers

a. ProbeOn Plus slides, MicroProbe work station, Fisher Scientific, Pittsburgh, PA.
b. Automation buffer, Biomeda Corp., Foster City, CA.
c. Genius^™ 5 kit, Boehringer Mannheim, Indianapolis, IN.

References

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