Improved Diagnosis of Psittacine Viral Diseases with DNA in situ Hybridization

Raymond P. Campagnoli and Kenneth S. Latimer

Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602-7388 (USA)

Abstract: DNA probes are especially useful in a diagnostic setting where suspected viral disease is present but examination of H&E-stained tissues is equivocal, or when poor specimens are submitted for histological diagnosis. Several cases are presented where DNA in situ hybridization was used to provide a definitive diagnosis of viral infection in various psittacine birds.

Key words: DNA, Psittacine, in situ Hybridization, Polymerase chain reaction, Adenovirus, Herpesvirus, Polyomavirus, Psittacine beak and feather disease virus, Pacheco’s parrot disease, Virus, Viral diagnostics  

Introduction

Viruses are a major cause of morbidity and mortality in companion and aviary psittacines. ¹ Polyomavirus, psittacine beak and feather disease virus (PBFDV), Pacheco’s disease virus, and adenovirus can all produce nuclear inclusions in affected tissues. In early stages of viral infection, typical viral inclusions may be absent or look similar in each of these diseases. The appearance of typical viral inclusions, distribution of lesions, and medical history can often yield a definitive disease diagnosis; however, a number factors can lead to an incorrect or incomplete diagnosis based upon examination of H&E-stained tissue sections alone. DNA in situ hybridization (ISH) has been used to diagnose these four common viral diseases of psittacine birds. ²

Samples submitted for histopathology are sometimes less than ideal. With a prolonged postmortem interval, autolysis may obscure viral inclusions, while nuclear edema may mimic nuclear inclusions. Tissues that have been frozen before fixation can have significant cellular disruption. Often the desired tissue(s) to document viral infection are not submitted or may be subject to poor fixation, mechanical damage, or other forms of mishandling. Occasionally, the biopsy specimen may be too small to provide a definitive diagnosis based upon examination of H&E-stained sections. Alternatively, biopsy or necropsy specimens taken from birds in the early stages of viral infection may lack characteristic inclusions or tissue lesions. Another problem is that concurrent viral infections may be present in the patient. Lastly, inclusions of undetermined significance have been observed in avian tissues³ that may be misdiagnosed as herpesvirus, polyomavirus, or adenovirus infections.

In these instances, DNA ISH has been used to provide a definitive diagnosis of viral infection and to detect concurrent viral infections. This manuscript demonstrates the usefulness of this technique in diagnosing viral infections in psittacine birds, especially when H&E-stained tissue sections are evaluated with equivocal results.

Material and Methods

Case materials and birds: The case material was selected from submissions by in-house clinicians, practicing veterinarians, and avian researchers. All birds had naturally acquired viral infections. One biopsy was from a live bird and the other tissue specimens were collected at necropsy.   All specimens were fixed in 10% neutral-buffered formalin, routinely processed, embedded in paraffin, and sectioned at a thickness of 3µm. The birds included: 1 Eclectus Parrot (Eclectus roratus vosmaeri), 1 African Grey Parrot (Psittacus erithacus), 1 Budgerigar (Melopsittacus undulatus), 1 White-bellied Caique (Pionites leucogaster), 1 Lovebird (Agapornis sp. ), and 1 Ornate Lorikeet (Trichoglossus ornatus).

In situ hybridization: ISH procedures were performed using published procedures. ⁴ A MicroProbe^a work station was used to handle the slides and regulate temperatures. Briefly, 3 µm paraffin embedded tissue sections were placed on ProbeOn Plus^a slides. The slides were dewaxed with limonene and xylenes and rehydrated through graded ethanols to Automation Buffer. ^b The tissues were digested in 0.3% pepsin in Automation Buffer (pH 2) for 10 minutes and heated to 105°C to inactivate the pepsin. The slides subsequently were pre-hybridized in 100% deionized formamide for 5 minutes at 105°C. Digoxigenin end-labeled oligonucleotide probes, at a concentration of 10 to 20 nM in hybridization buffer (22.5% deionized formamide, 7.5% chondroitin sulfate, 5 x SSC, 50 mM phosphate buffer, and 0.25% Blocking reagent^c), were placed on the slides for 5 minutes at 105°C and incubated for an additional hour at 37°C. PBFDV (probe FN8), polyomavirus probes (FN19 and FN40), Pacheco’s disease virus (probe FN65), and adenovirus (probe FN96) probes were used. In addition a PCR-generated polyomavirus probe (VP1) was used for confirmation of viral infection in some cases.

All tissue sections were hybridized to at least two different oligonucleotide probes, so that one probe could act as a negative control. The slides were washed in 0.5 x SSC, 0.4% Tween 20 and 0.25% Brig 35 for 5 minutes, and in 0.2 x SSC, 0.4% Tween 20 and 0.25% Brig 35 at 37°C for 5 minutes. Anti-digoxigenin^c antibody conjugated to alkaline phosphatase was used as the indicator system. The antibody was used at a dilution of 1:500 in a mixture of 100 mM tris pH 7.5, 150 mM sodium chloride, and 0.3% Triton X-100. The diluted antibody subsequently was placed on the slides and incubated for 1 hour. Nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate in 100 mM tris pH 9.5, 100 mM sodium chloride, 50 mM magnesium chloride, 0.4% Tween 20, and 0.25% Brig 35 was placed on the slides for 2 hours at 37°C. The slides were rinsed in deionized water, counterstained in 1% fast green FCF, dehydrated through graded ethanols, coverslipped, and examined by light microscopy.

Polymerase chain reaction (PCR) procedures: Viral infection subsequently was confirmed in several of the birds by PCR of target viral DNA extracted from paraffin-embedded tissues. Either the method was as described previously,⁴ or the nucleic acid was extracted by adding 200 µl 1% Triton X-100 to a 15 µm paraffin section in a microfuge tube which was incubated at 95°C for 1 hour. Five µl of this sample was used for the PCR incubation. A psittacine-specific sequence of cytochrome B was chosen as a reporter target to monitor the presence of nucleic acid and the absence of PCR inhibitors in the extraction mix.

Results

Case #1, Eclectus Parrot (Eclectus roratus vosmaer): The bird died suddenly and only formalin fixed liver was submitted for histological examination. Microscopic lesions included multifocal to confluent hepatocellular necrosis. Scattered hepatocytes contained variable-sized, circular, intracytoplasmic vacuoles suggestive of lipid accumulation (Fig. 1A). Although occasional hepatocyte and Kupffer nuclei appeared edematous, typical polyomavirus inclusions were not observed in H&E-stained tissue sections. Following DNA ISH, polyomavirus nucleic acid was observed within the nuclei of Kupffer cells, stromal cells, and a few hepatocytes (Fig. 1B). The final diagnosis was avian polyomavirus infection.

Fig. 1A. Eclectus Parrot, liver, H&E stain. Hepatocellular necrosis is present.	Fig. 1B. Eclectus Parrot, liver, ISH for polyomavirus. Polyomavirus nuclear inclusions stain blue.

Case #2, African Grey Parrot (Psittacus erithacus), male, 3-year-old: A small piece of formalin fixed liver was submitted by mail in a plastic bag inside a standard business envelope. The tissue was flattened and fragmented during cancellation by the post office. In addition, the liver was severely autolytic prior to fixation. Despite crush artifact and severe autolysis, there was evidence of pre-existing hepatocellular necrosis and glassy intranuclear inclusions in the H&E-stained tissue sections (Fig. 2A). DNA ISH confirmed the presence of Pacheco’s disease virus nucleic acid within the inclusions (Fig. 2B). The definitive diagnosis was Pacheco’s parrot disease.

Fig. 2A. African Grey Parrot, mechanically damaged and autolytic liver, H&E stain. Glassy nuclear inclusions are present (center).	Fig. 2B. African Grey Parrot, mechanically damaged and autolytic liver, ISH for Pacheco’s disease virus. Nuclear inclusions stain blue confirming the presence of Pacheco’s disease virus DNA within inclusions.

Case #3, Budgerigar (Melopsittacus undulatus), male: Scattered renal tubular epithelial cells contained large, glassy, basophilic nuclear inclusions. On H&E-stained tissue sections, these inclusions were suggestive of polyomavirus or adenovirus infection (Fig. 3A). This bird was negative for polyomavirus, adenovirus, Pacheco’s disease virus, and PBFDV (Fig. 3B) nucleic acids by DNA ISH using oligonucleotide probes. Additional probing with a PCR- generated probe from the VP1 region of avian polyomavirus also was negative. This case also was negative for polyomavirus when tested by PCR. The "inclusions" were not identified specifically.

Fig. 3A. Budgerigar, kidney, H&E stain. Renal tubular epithelial cells contain large intranuclear inclusions.	Fig. 3B. Budgerigar kidney, ISH for polyomavirus. Lack of blue staining in large intranuclear inclusions indicates absence of polyomavirus DNA.

Case #4, White-bellied Caique (Pionites leucogaster), adult: Microscopically, the feathers were characterized by necrosis, hemorrhage, and heterophilic infiltrates. Some feathers contained variably-sized, gray, nuclear inclusions (Fig. 4A). Similar inclusions also were present within the spleen and glomeruli. Necrosis was observed in both the spleen and liver; however, inclusions were not observed in the liver. Use of DNA probes confirmed the presence of polyomavirus nucleic acid (Fig. 4B), but did not detect PBFDV nucleic acid. This bird also was PCR positive for polyomavirus and PCR negative for PBFDV. The lesions in this bird are not unusual; however, polyomavirus infection rarely has been associated with clinical disease in adult psittacine birds. The death of this bird was attributed to polyomavirus infection.

Fig. 4A. White-bellied Caique, spleen, ISH for polyomavirus. Blue stain in inclusions indicates the presence of polyomavirus DNA (center).	Fig. 4B. White-bellied Caique, spleen, ISH for PBFDV. Lack of blue staining indicates the absence of PBFDV DNA.

Case 5#, Lovebird (Agapornis sp.): Sections of the liver had multifocal hepatocellular necrosis, bile duct hyperplasia, hemosiderosis, mild fibrosis, and aggregates of degenerating thrombocytes. Sections of the spleen revealed hemosiderosis, necrosis, and gray intranuclear inclusions suggestive of polyomavirus infection (Fig. 5 A&B). Similar inclusions also were observed within glomerular tufts. Skin sections contained developing feathers with necrosis, hemorrhage, and infrequent PBFDV-like, basophilic inclusions. The inclusions were nuclear in feather epithelial cells and cytoplasmic in macrophages. DNA ISH document combined polyomavirus and PBFDV nucleic acid within the submitted tissues (Fig. 5 C&D). In addition, tissues from this bird were positive for both viruses by PCR. The definitive diagnosis was concurrent polyomavirus and PBFDV infections.

Fig. 5A. Lovebird, feather follicle, ISH for polyomavirus. Blue staining inclusions indicate the presence of polyomavirus DNA.	Fig. 5B. Lovebird, feather follicle, ISH for PBFDV. Blue staining indicates the presence of PBFDV DNA.

Fig. 5C. Lovebird, spleen, ISH for polyomavirus. Blue staining intranuclear inclusions indicate the presence of polyomavirus DNA.	Fig. 5D. Lovebird, spleen, ISH for PBFDV. Blue staining indicates the presence of PBFDV DNA.

Case #6, Ornate Lorikeet (Trichoglossus ornatus), male, neonate: In this case a very small portion of skin with a single feather follicle was submitted for histological examination. Mild, multifocal infiltrates of lymphocytes and plasma cells were present around dermal blood vessels (Fig. 6A). Neither nuclear nor cytoplasmic inclusions were observed. However, DNA ISH was positive for PBFDV nucleic acid (Fig. 6B) but was negative for polyomavirus nucleic acid (Fig. 6C). The definitive diagnosis was PBFDV infection.

Fig. 6A. Ornate Lorikeet, feather follicle, H&E stain. Mild epithelial cell necrosis is present.	Fig. 6B. Ornate Lorikeet, feather follicle, ISH for PBFDV. Blue staining indicates the presence of abundant PBFDV DNA.	Fig. 6C. Ornate Lorikeet, feather follicle, ISH for polyomavirus. Lack of blue staining indicates the absence of polyomavirus DNA. Note melanin pigment granules in the feather.

Discussion

The cases presented herein had an equivocal diagnosis of viral infection based upon the examination of H&E-stained tissue sections alone. The use of viral-specific DNA probes allowed definitive diagnoses of viral infection to be made in 5 of 6 birds. Although our experience indicates that PCR can be more sensitive than ISH in detecting avian viruses,⁴ the PCR results agreed with those of ISH in the cases presented here. Furthermore, DNA ISH is less subject to false positive test results than is PCR testing.

Tissues from the Eclectus Parrot (Case #1) and Ornate Lorikeet (Case #6) lacked typical viral inclusions on H&E-stained tissue sections. However, DNA ISH using probes FN19 and FN40 readily demonstrated the presence of avian polyomavirus infection.

The liver specimen from the African Grey Parrot (Case #2) was autolytic and severely macerated. Despite these artifacts, DNA ISH still provided a definitive diagnosis of Pacheco’s parrot disease.

Based upon examination of H&E-stained sections, tissues from the Budgerigar appeared to have viral inclusions. However, these tissues were negative for adenovirus, herpesvirus, polyomavirus, and PBFDV nucleic acids. In addition, these tissues were negative for avian polyomavirus nucleic acid using the oligonucleotide probes (FN19 and FN40) and the long, double-stranded, PCR-generated probe (VP1). This VP1 probe will detect different strains of avian polyomavirus in blue-bills,⁵ seedcrackers,⁵ canaries, finches, and psittacine birds. Therefore, DNA ISH excluded a diagnosis of avian polyomavirus in this Budgerigar. More recently, these tissues also have been shown to be free of discernable virions by transmission electron microscopy. This case investigation underscores the importance of making a definitive diagnosis of viral infection by techniques other than examination of H&E-stained tissue sections alone. In some instances, the pathologist may be misled by the presence of pseudoinclusions. To date, we have seen at least 7 to 8 psittacine birds with unidentifiable renal tubular nuclear "inclusions" in H&E-stained sections of kidney.

The White-bellied Caique (Case #4) demonstrates that polyomavirus infection alone can be fatal in naive, adult, psittacine birds. Other researchers have suggested that adult psittacines are severely affected by polyomavirus only when they are immunocompromised by concurrent PBFDV infection,⁶ but that is shown not to be the case here.

Finally, DNA ISH was used to confirm concurrent infection with polyomavirus and PBFDV in a Lovebird (Case #5). Birds with PBFDV may be immunosuppressed and it is important to check for other viruses or other disease organisms when PBFDV is diagnosed. This bird was PCR positive for polyomavirus and for PBFDV. Although our experience indicates PCR can be more sensitive than ISH in detecting avian viruses,⁴ in these cases the PCR results agreed with those from ISH.

In summary, these cases illustrate the usefulness of DNA ISH in diagnosing certain avian viral diseases. In some instances, a definitive diagnosis may be obtained that would be difficult or impossible to achieve by examination of H&E-stained tissue sections alone. ISH is very satisfying to use when the result definitively shows the virus where you expect to find it and sometimes where you do not expect to find it.

Sources and Manufacturers

^aFisher Scientific, Pittsburgh, PA 15238 (USA)
^bBiomeda, Foster City, CA 94404 (USA)
^cBoehringer Mannheim Biochemicals, Indianapolis, IN 46250 (USA)

References

1. Ritchie BW: Avian Viruses: Function and Control. Wingers Publishing, Inc. Lake Worth, FL, 1995.

2. Ramis A, Latimer KS, Niagro FD, Campagnoli RP, Ritchie BW, Pesti D: Diagnosis of psittacine beak and feather disease (PBFD) viral infection, avian polyomavirus infection, adenovirus infection and herpesvirus infection in psittacine tissues using DNA in situ hybridization. Avian Pathol 23:643-657, 1994.

3. Cardona CJ, Trampel DW, Bickford AA, Daft BM, Droual R, Kinde H: Intranuclear inclusions of unknown pathogenic significance from diagnostic cases in three avian species. Avian Dis 37:244-253, 1993.

4. Latimer KS, Niagro FD, Campagnoli RP, Ritchie BW, Pesti DA, Steffens WL: Diagnosis of concurrent avian polyomavirus and psittacine beak and feather disease virus infections using DNA probes. J Assoc Avian Vet 7:141-146, 1993.

5. Garcia AP, Latimer KS, Niagro FD, Norton TM, Campagnoli RP, Harmon BG, Howerth EW, Ritchie BW: Diagnosis of polyomavirus infection in seedcrackers (Pyrenestes sp. ) and blue bills (Spermophaga haematina) using DNA in situ hybridization. Avian Pathol 23:525-537, 1994

6. Phalen DN: Avian Polyomavirus: My Thoughts. http://www.blackstone-aviaries.com/polyom.html

This Page Last Updated May 15, 1998

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