Characterization of a Herpesvirus Associated with Tracheitis in Gouldian Finches (Erythrura [Chloebia] gouldiae)

Abstract

Severe tracheitis and bronchitis were identified in two fatal cases of respiratory disease affecting a flock of Gouldian finches (Erythrura [Chloebia] gouldiae). Intranuclear inclusion bodies in epithelial cells of the upper respiratory tract were identified in samples from two birds. Electron microscopic examination showed that the inclusions consisted of viral particles consistent in appearance with Herpesviridae. Degenerate PCR primers targeting a conserved region of the herpesviral-DNA-dependent DNA polymerase were used to amplify a region of DNA isolated from tissues with lesions from each animal. Nucleotide sequencing of the PCR products yielded identical viral sequences that were distinct from known herpesviruses. An analysis of sequence homology indicated that these gene segments appear to belong to a member of the subfamily Alphaherpesvirinae.

The Gouldian finch (Erythrura [Chloebia] gouldiae) is an endangered passerine bird native to northern Australia. Wild Gouldian finch populations have been declining markedly, as illustrated by a 70% reduction of this species between 1972 and 1982 (20). In 1996, the wild population was estimated to be 2,000 individuals (19). The Gouldian finch is also highly prized in aviculture. Because of this value, it is commonly presented to avian veterinarians for diagnosis and treatment.

The Herpesviridae are a diverse family of enveloped, double-stranded-DNA viruses found in many different orders of vertebrates, including fish, amphibians, reptiles, birds, and mammals (14). Herpesviruses have been further divided into the subfamilies Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae, which were initially based on properties of infection, host range, and behavior in culture. Phylogenetic relationships of herpesviruses are now formally based on genetic content, as defined by homology of nucleic acid sequences and identification of particular genes unique to a virus subset (15). DNA evidence has largely reinforced the extant classification system and suggests the presence of further subfamilies. All known sequences of fish and amphibian herpesviruses appear to be distinct from the above-named subfamilies, and an oyster herpesvirus may also be distinct (14). All currently available sequence data derived from avian herpesviruses have been consistent with the subfamily Alphaherpesvirinae (14, 21).

Herpesviruses of passerine birds have been identified in finches, weavers, starlings, canaries (13), and cardinals (9). In Gouldian finches, herpesviruses have been identified by electron microscopy in disease outbreaks in captive flocks in Belgium (5), Switzerland (23), and Austria (18). All three outbreaks were associated with high mortality, and affected animals had respiratory disease, with viral inclusions identified in their respiratory epithelia. Electron microscopic examinations of tissues from the birds in one outbreak identified a virus that was considered to be a cytomegalovirus based on ultrastructural appearance (5). In the study described here, we investigated the potential infectious etiology of an outbreak of respiratory disease in Gouldian finches. Characteristic cytopathic lesions, ultrastructural appearance, and sequence analysis of a novel gene segment identified a herpesvirus. This outbreak closely resembled outbreaks previously seen with Gouldian finches in Europe and provided additional evidence for a herpesvirus associated with disease in passerine birds.

MATERIALS AND METHODS

Index cases.

Three Gouldian finches were brought to the Veterinary Teaching Hospital at the University of Guelph for evaluation of labored breathing. In the aviary from which the birds originated, two Gouldian finches had died the morning of presentation and two additional Gouldian finches were showing signs of dyspnea. The aviary contained 20 Gouldian finches of various color mutations. Pairs of pin-tailed parrotfinches (Erythrura prasina) and double-barred finches (Taeniopygia bichenovii) had been introduced into the aviary 1 month earlier and were housed in separate cages. Dyspnea in the Gouldian finches was first noted 1 day prior to presentation. The birds were eating well but previously had mild diarrhea. The birds' plumage had been puffed up, and a clicking noise had been noted during respiration. The birds were housed in a pen with several other finches and were not receiving any medications. Upon presentation, the three live finches appeared to have labored breathing. Loose stools were noted in the carrier. Hepatomegaly was apparent on physical examination of one of the finches. The two recently deceased finches were submitted for postmortem examination. All live dyspneic Gouldian finches were treated orally with 60 mg of trimethoprim-sulfamethoxazole (Novo-trimel; Novopharm, Toronto, Ontario, Canada) per kg of body weight twice daily for 21 days.

Pathology and electron microscopy.

A systematic necropsy with selection of tissues for microscopic examination was performed. Select tissues were immersed in buffered formalin for 24 h, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. For ultrastructural examination, sections of formalin-fixed trachea were placed in 2.5% glutaraldehyde in 0.2 M phosphate buffer at pH 7.4 for 48 h. After three washes in phosphate buffer, these samples were postfixed in osmium tetroxide in the same buffer at 4°C for 3 h, dehydrated by immersion in increasing ethanol concentrations (70 to 100%), cleared twice for 15 min in propylene oxide, and infiltrated for 1 h in equal volumes (1:1) of propylene oxide and epon at room temperature. The samples were then embedded in pure epon, and ultrathin sections on grids were stained with 5% uranyl acetate in 50% ethanol for 20 min and in 5% lead citrate.

PCR amplification, sequencing, and analysis.

DNA was extracted from lung tissue collected during necropsy. DNA was extracted (DNeasy tissue kit; QIAGEN, Valencia, Calif.), and a region of the herpesvirus DNA-dependent DNA polymerase was amplified as previously described (21). Three separate DNA extractions and amplifications were performed for each bird. The final PCR products were separated by electrophoresis in 1% agarose gels, and the bands were cut out of the gel with sterile scalpel blades. The DNA was purified from the agarose plugs (QIAquick PCR purification kit; QIAGEN) and sequenced (Molecular Supercentre, University of Guelph, Guelph, Ontario, Canada) in both directions.

The sequences were compared to known sequences in the GenBank (National Center for Biotechnology Information, Bethesda, Md.), EMBL (Cambridge, United Kingdom), and Data Bank of Japan (Mishima, Shiuoka, Japan) databases using TBLASTX (1). Predicted protein sequences of homologous 55- to 61-amino-acid segments of herpesvirus DNA polymerases were aligned by the Jotun Hein algorithm (11) using MegAlign (DNAstar, Madison, Wis.). A phylogenetic tree was constructed using the PHYLIP 3.573c package (6). Trees were outgroup rooted using DNA polymerase delta 1 of Mus musculus (GenBank accession no. BC009128). Gaps of all lengths were counted as single events. One hundred bootstrapped data sets were created. Each data set was used 10 times in a random order of input to create the most probable trees, and a majority rule consensus tree was then created.

Nucleotide sequence accession number.

The avian sequence was identified here was submitted to GenBank (accession no. AF520812).

RESULTS

Index cases.

The postmortem examination of the first finch revealed a dark red area 3 mm in diameter affecting the dura of the frontoparietal region of the skull. The lungs and trachea appeared grossly normal. On microscopic examination, it was observed that the pseudostratified epithelium of the trachea had areas of hyperplasia and variable erosion with superficial sloughing of necrotic epithelial cells. The tracheal lumen was filled with degenerate heterophils, macrophages, necrotic sloughed epithelial cells, fibrin, cellular debris, and large numbers of bacterial colonies. Tracheal epithelial cells showed karyomegaly, with nuclei being 20 to 30 μm in diameter and containing large, intensely basophilic inclusion bodies (Fig. 1). Large numbers of plasma cells and lymphocytes had infiltrated the lamina propria. The epithelium of the bronchi was hyperplastic, and the bronchial epithelial cells had similar inclusions, with sloughing of affected cells. The lung was markedly congested. The liver had multifocal areas of hematopoiesis. Scattered apoptotic hepatocytes were present. In the kidney, the mesangium of the glomerular tufts was markedly thickened due to the deposition of a large amount of intensely eosinophilic amorphous material consistent in appearance with amyloid. No significant lesions were seen in the remaining tissues.

FIG. 1.

Section of the tracheal epithelium from a Gouldian finch with respiratory disease. Dark nuclear inclusions surrounded by a halo (arrow) are present throughout the epithelial cells lining the trachea. Necrotic epithelial cells are sloughing into the apical lumen of the trachea (arrowhead).

Gross examination of the second finch revealed no abnormalities. However, on histological examination, the trachea and bronchi showed lesions similar to those of the first bird. Multifocal accumulations of macrophages with abundant granular cytoplasm were present in the lung, particularly in perivascular areas, liver, myocardium, and epicardium at the base of the heart. On Ziehl-Neelsen staining, these macrophages were observed to contain large numbers of acid-fast bacteria. No significant lesions were seen in the remaining tissues.

In total, four additional birds were lost in the aviary in the week following presentation: three Gouldian finches and a pin-tailed parrotfinch. All Gouldian finches showed signs of respiratory distress. Postmortem examinations were not performed on these birds. Thus, this represented a mortality rate of 25% and a morbidity rate of approximately 50%. After 1 week, no further clinical signs were noted by the owner. No further losses were seen in the aviary over the next 4 months.

Electron microscopy.

Ultrastructural examination of the tracheal epithelium showed that the nuclear inclusions observed on light microscopy consisted of dense aggregates of viral particles (Fig. 2a). Viral particles were mainly in the nucleus, with fewer particles in the immediate perinuclear and cytoplasmic areas. Enveloped capsids as well as empty envelopes were apparent (Fig. 2b). The size of the particles was approximately 150 nm, and they were present throughout the affected epithelial tissues.

FIG. 2.

Electron micrographs of tracheal epithelial cells from a Gouldian finch with respiratory disease. (a) The nucleus contains large numbers of viral particles with various electron densities. Some particles are present in the perinuclear area and throughout the cytoplasm. Bar = 1 μm. (b) The viral particles are approximately 150 nm in diameter and consist of enveloped viral particles with an electron-dense core (capsid) or an electron-lucent core (empty envelopes). Bar = 200 nm.

PCR amplification and sequence.

Amplification of the Gouldian finch herpesvirus DNA polymerase resulted in a product of 175 bp when primer sequences were excluded. Sequenced PCR products from the two finches were 100% homologous with each other.

TBLASTX analysis of the Gouldian finch herpesvirus DNA polymerase sequences showed the highest homology with gerrhosaurid herpesvirus 3 DNA polymerase (GenBank accession no. AF416630). The majority-rule consensus phylogenetic tree of predicted protein sequences of homologous 55- to 61-amino-acid segments of herpesvirus DNA polymerases is shown (Fig. 3). Although the division between the alphaherpesviruses and the betaherpesviruses did not have a bootstrap value over 50, Gouldian finch herpesvirus clustered with gallid herpesvirus 1 (infectious laryngotracheitis virus of chickens), which has had multiple genes sequenced and was shown to be an alphaherpesvirus by sequence homology (12). Infectious laryngotracheitis virus of chickens has been further subclassified within the subfamily Alphaherpesvirinae as genus 04 (14). These results suggest that this Gouldian finch virus belonged in a closely related genus of the subfamily Alphaherpesvirinae. However, more sequencing of the genome will be required to confirm this preliminary group assignment. Based on naming conventions, this virus should be named passerid herpesvirus 1 (17).

FIG. 3.

Phylogenetic tree of predicted protein sequences of homologous 55- to 61-amino-acid segments of herpesviral DNA polymerases. Trees were outgroup rooted using DNA polymerase delta 1 of M. musculus (GenBank accession no. BC009128). Bootstrap values are shown. Branchings with bootstrap values less than 50 are not shown, and areas where these branchings occurred are shown as heavy lines. Homologous polymerases (GenBank accession numbers are given in parentheses) were from bovine herpesvirus 2 (HV2) (AAD55134), columbid HV1 (AAD30145), equid HV1 (AAB02465), equid HV2 (P52367), felid HV1 (CAA12264), gallid HV1 (AAD56202), gallid HV2 (AAA79862), gerrhosaurid HV1 (AF416628), gerrhosaurid HV3 (AF416630), human HV1 (P09854), human HV3 (BAB41073), human HV4 (DJBE2L), human HV5 (AAG02102), human HV6 (AAD49652), human HV7 (T41940), human HV8 (AAC57974), leoporid HV2 (AAC55655), mandrill cytomegalovirus (CMV) (AAG39064), meleagrid HV1 (AAG30070), olive ridley turtle HV (AAC26684), passerid HV1 (AF520812), psittacid HV1 (AAC55656), saimiriine HV2 (AAA46165), tortoise HV (BAB40430), tupaia HV1 (Q9YUS3).

DISCUSSION

Respiratory disease in passerine birds has been associated with many causes and is frequently associated with stress. Infectious causes of dyspnea include the mite Sternostoma tracheacolum (20), various bacteria (16), coccidia (8), herpesvirus (5), and poxvirus (2). Several viruses have been implicated as causative or contributory to respiratory disease in Gouldian finches, including a polyomavirus-like organism identified by electron microscopy of lung tissue (7) and an eastern equine encephalitis virus isolated from a Gouldian finch with dyspnea (4). Herpesviruses cause upper respiratory tract disease in other birds and were considered likely candidates for the outbreak in Gouldian finches identified here because the clinical picture and pathological findings were similar to those of previous herpesvirus infections reported for this species in Europe (5, 18, 23). All previous outbreaks were associated with respiratory disease and mortality, and all were seen in mixed-species flocks with the recent introduction of new birds to the aviary. However, the 25% mortality seen in Gouldian finches in this outbreak was lower than the 70 to 100% mortality associated with two of the previously documented outbreaks (5, 23). Also, the conjunctivitis observed in two previous outbreaks (5, 23) was not identified in this flock. In the remaining report, affected birds presented with respiratory signs without conjunctivitis (18), as was seen in this outbreak. The mortality rate was not provided. Organisms that stained with acid-fast stain were identified in the macrophages of several organs in one bird in this report. The pathogenesis of infection with mycobacterial organisms is typically chronic, insidious, and associated with persistent macrophage infection and dysfunction. Mycobacterium spp. were therefore considered unlikely agents of the rapid disease course and epithelial lesions observed in this case, although chronic infection with Mycobacterium spp. may be associated with immunosuppression and therefore may have rendered one of the birds in this report more susceptible to an overwhelming herpesviral infection.

Confirmation of a herpesvirus as the agent associated with respiratory disease in this group of birds was attempted by electron microscopy and gene amplification. The initial light microscopic lesions observed were highly suggestive of the cytopathic effects associated with alphaherpesviral infection in other species, including gallid herpesvirus 1 (infectious laryngotracheitis). Further ultrastructural examination of affected tissues confirmed the presence of a virus of an appropriate size and morphology for the Herpesviridae. While being fairly species specific, Alphaherpesvirinae typically exhibit a more varied host range than Betaherpesvirinae or Gammaherpesvirinae and can establish latent infections in sensory ganglia (17). Nucleic acid sequence analysis suggested that the viruses in the affected tissues of the Gouldian finches were most closely related to the alphaherpesviruses. While these findings are consistent with the clinicopathological findings, only 175 bp of a genome likely exceeding 100,000 bp was analyzed. Thus, while the sequences obtained are strongly suggestive of a novel herpesvirus associated with the lesions in these birds, it would be desirable to confirm these findings with amplification of a larger region of the viral genome. Alternatively, definitive pathogenic properties of the virus would be best demonstrated by virus isolation in tissue culture and reinfection of naïve birds. Unavailability of naïve birds from the species limited this approach.

Classification of the avian herpesviruses appears to be most affected by reclassification according to genetic homology. Based on nucleic acid sequence analysis, gallid herpesvirus 2 (Marek's disease virus) and meleagrid herpesvirus 1 (herpesvirus of turkeys) have been reclassified from gammaherpesviruses to alphaherpesviruses (3, 17). This new classification scheme places all known characterized avian herpesviruses into the alpha subfamily.

Sequence analysis of the herpesviruses of birds should contribute to further understanding of viral phylogeny. Phylogenetic analysis of mammalian herpesviruses suggested that many elements in the branching patterns of Herpesviridae are congruent with branching patterns for the corresponding host species (14). The branching of the Alphaherpesvirinae from the Betaherpesvirinae and the Gammaherpesvirinae was tentatively estimated to have occurred approximately 200 million years ago (14). According to molecular evidence, after the divergence of squamates from other reptiles and birds, the ancestors of birds diverged from the lineage leading to turtles and crocodilians (10). The divergence of birds from turtles and crocodilians was estimated by molecular phylogeny to have occurred approximately 228 million years ago, although the oldest avian fossils date from only approximately 147 million years ago (10). The divergence of Neoaves, containing passerine birds, from Galloanserae, containing galliform birds, was estimated to have occurred approximately 104 million years ago (22). Nongalliform birds likely harbor other as-yet-unknown herpesviruses from diverse host taxa. The discovery and characterization of these viruses may yield insight into viral as well as vertebrate evolution.