Psittacid herpesvirus 3 infection in rose-ringed parakeets in southern Brazil

Abstract

We diagnosed disease caused by psittacid herpesvirus 3 (PsHV-3), a novel psittacid pathogen, in rose-ringed parakeets (Psittacula krameri) housed in an exotic psittacine breeding colony in southern Brazil. The disease affected several adult birds. Clinical signs included apathy, tachypnea, and wheezing. Four birds were autopsied, and sections of lungs and liver were examined histologically and by electron microscopy (EM), revealing pulmonary congestion, bronchopneumonia, or multifocal necrosis of tertiary bronchi, with syncytial cells and eosinophilic intranuclear inclusion bodies. Viral particles morphologically compatible with herpesviruses were observed by EM in lung sections. PCR with pan-herpesvirus primers performed on total DNA extracted from paraffinized tissue resulted in a 278-bp product. Sequencing of the amplicon revealed 93% nucleotide identity with a PsHV-3 sequence available in GenBank. Phylogenetic analysis grouped the obtained sequence with the only PsHV-3 DNA polymerase gene sequence available (GenBank accession JX028240) and separated the sequence from psittacid herpesviruses 1 and 2. The clinical, pathologic, and molecular findings support the association of PsHV-3 with pneumonia found in these rose-ringed parakeets in southern Brazil.

Herpesviruses are important pathogens associated with several diseases in avian species, including infectious laryngotracheitis and Marek disease in domestic poultry² and Pacheco disease in psittacine birds.⁹ To date, 3 herpesviruses have been identified among the birds of the Psittacidae family: psittacid herpesvirus 1, 2, and 3 (PsHV-1, -2, and -3)^6,8 of which only PsHV-1 (Psittacid alphaherpesvirus 1) has been officially recognized as a viral species. These viruses belong to the family Herpesviridae, subfamily Alphaherpesvirinae, genus Iltovirus (https://talk.ictvonline.org/taxonomy/). PsHV-3 was first detected during an outbreak of a respiratory disease in Bourke parrots (Neopsephotus bourkii) in the United States,⁶ and, subsequently, detected only once among eclectus parrots (Eclectus roratus) in Australia.²

In an ornamental nursery in the central region of Rio Grande do Sul, in southern Brazil, ~ 40 birds of the Psittacidae family were housed as a group in a nursery of ~ 25 m³. Several rose-ringed parakeets (Psittacula krameri) died over 4 wk; the exact number was not provided by the owner. Between August and September 2013, an adult female (case 1), a young male (case 2), and 2 adult males (cases 3, 4) were found dead. A few days before the deaths, the owner had noticed that the female was walking on the ground, being chased by the other birds. One of the adult males was depressed, tachypneic, and was wheezing.

The 4 dead psittacines were sent to the Central Laboratory of Diagnosis of Avian Pathology of the Federal University of Santa Maria, Rio Grande do Sul, Brazil (LCDPA-UFSM) for autopsy. External examination revealed poor (cases 1, 2) to normal (cases 3, 4) nutritional status, brittle feathers, dirty tails, and feces around the cloaca of all birds. Macroscopically, pulmonary congestion was observed in cases 1–3, as well as lesions compatible with pneumonia, such as yellow firm nodules (case 2), small gray nodules (case 3), or light-yellow areas spread over the pulmonary parenchyma (case 4; Fig. 1). The liver was mottled with light and dark-red areas and was enlarged with rounded edges. The wall of the crop of case 4 was white and very thick. The intestines of all birds had congested vessels and hemorrhagic luminal content. In a previous case of PsHV-3 infection, the macroscopic lesions observed at autopsy were moderate congestion, edematous lungs, and small amounts of fibrinous exudate in the air sacs and tracheal lumen.⁶ We found no changes in the trachea or air sacs in our cases.

Figure 1.

Pale yellow nodules (arrows) in the pneumonic lungs in case 4, an adult male rose-ringed parakeet.

Tissue sections from the lungs and liver of all birds, and crop and spleen of case 4, were fixed in 10% formalin and processed routinely. Histologically, the main lesion consisted of mild-to-severe multifocal necrosis of the epithelium of secondary and tertiary bronchi. Eosinophilic intranuclear inclusion bodies were observed within the atrial respiratory epithelial cells of the lungs (Fig. 2A), including individual epithelial cells and multinucleate syncytial cells, which were observed in the walls of secondary bronchi, tertiary bronchi, and air capillaries. Inclusion bodies usually occupied the entire nucleus or were surrounded by a thin clear halo. Some inclusion bodies were lightly basophilic. There was also fibrin in the lumen of tertiary bronchi, and severe congestion and multifocal hemorrhage in the lung parenchyma. In the liver in case 1, a few hepatocytes had nuclei with marginated chromatin and contained eosinophilic inclusion bodies surrounded by a thin clear halo. Inclusion bodies in the epithelial cells of air sacs, next to the lung, were observed in case 3. In the spleen in case 4, there were small necrotic areas with fibrin deposition and cells with eosinophilic intranuclear inclusion bodies adjacent to these areas. The lesions that we noted are similar to those found in birds infected with PsHV-3 in other studies, which included necrosis of the respiratory epithelium, bronchopneumonia of various severity levels, and airsacculitis with syncytial cells containing eosinophilic viral inclusion bodies.⁶ The bronchopneumonia and necrotizing peribronchitis observed in our cases were associated with the presence of syncytial cells, most of which contained intranuclear eosinophilic viral inclusion bodies, which are the characteristic of many herpesviral infections.²

Figure 2.

Pneumonia in a rose-ringed parakeet. A. Necrotizing bronchopneumonia associated with syncytial cells containing eosinophilic intranuclear inclusion bodies (arrow). H&E. 40×. B. Virions of ~ 145 nm diameter in the nucleus of a syncytial cell, with core, capsid, and envelope.

The herpesvirus virions contain envelope and capsid; their diameter can vary between 120 and 300 nm.¹ Examination by electron microscopy (EM) of the lung samples of 2 birds submitted for autopsy revealed the presence of virions in the intranuclear inclusion bodies in the syncytial cells. These viral particles, of ~ 145 nm diameter (Fig. 2B), are morphologically compatible with herpesvirus particles. Thus, the identification of herpesvirus-like virions in the lungs of affected birds suggested the association of herpesvirus with the development of the lesions.

To further pursue the identification of the viral agent associated with these lesions, total DNA extracted from paraffinized lungs was tested with a pan-herpesvirus PCR assay. DNA extraction was performed (QIAamp DNA FFPE tissue kit; Qiagen, Hilden, Germany), and the PCR assay used pan-herpesvirus degenerate oligonucleotide primers and PsHV-3–specific primers (both primers pairs targeted the viral DNA polymerase gene).^6,10 The PCR products were sequenced by the Sanger method, and the consensus sequence was obtained using the Staden program.⁷ The identity of these consensus sequences with sequences from GenBank was verified using BLAST software (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

The sample of case 4 was positive by PCR using a set of pan-herpesvirus primers, resulting in a 278-bp amplicon (GenBank accession MK922358); 220 bp of this amplicon had low or no homology with GenBank sequences, but 58 of the 278 bp had 93% homology with a unique PsHV-3 DNA GenBank polymerase gene sequence corresponding to the 3′ terminus of the DNA polymerase gene of PsHV-3 from GenBank (DNA pol gene positions 862 to 819 of the JX028240 sequence). A comparison of the complete amplicon sequence was not possible given the low number of PsHV-3 sequences deposited in GenBank. Thus, the obtained comparison and identity corresponded to ~ 20% of the JX028240 sequence.

The nucleotide identity of the obtained sequence of case 4 was significantly lower compared with other herpesviruses (74% identity with Passerid herpesvirus 1, AF520812), and no significant similarity was found with Psittacid herpesviruses 1 and 2. The lack of amplification in some samples may be related to degradation of genetic material given the long time in fixative and/or paraffin.

The 278 bp sequence obtained from case 4 was aligned with sequences of the DNA polymerase gene from 21 avian herpesviruses. The alignment was used to build a phylogenetic tree in MEGA X software⁴ using the maximum likelihood method with 1,000 bootstrap replicates (Fig. 3). The sequence of case 4 grouped with the sole/unique sequence of the PsHV-3 DNA pol gene available in GenBank (JX028240), and segregated from other psittacid herpesviruses, indicating that the amplified sequence belongs to this new herpesvirus species.

Figure 3.

Phylogenetic tree based on the nucleotide sequences of the DNA polymerase gene of avian herpesviruses. The equivalent gene of Felid alphaherpesvirus 1 (AJ224971) is used as the outgroup. The tree was constructed using the maximum likelihood method with 1,000 bootstrap replicates and implemented by MEGA X. Values > 70% are shown. The psittacid herpesvirus 3 sequence of the present report is identified with a black diamond.

The geographic distribution of PsHV-3 infection is probably broader than that which has been described to date. There are several reports of psittacines with macroscopic and microscopic lesions compatible with this infection, yet most were published before PsHV-3 gene sequencing.^2,6 Thus, these cases might not have been diagnosed correctly. There were reports of a “respiratory herpesvirus”, through macroscopic and microscopic evidence, in ring-necks imported from Australia to the United States,⁵ and in 14 ring-necks in Japan, brought from India. According to the authors, the herpesvirus was unknown at the time of publication.⁹ In addition, lung lesions similar to those observed in these cases have been described and associated with a “parakeet respiratory herpesvirus.”⁵

Another viral agent was also investigated in the same breeding birds. Psittacid circovirus (PsCV), which causes beak-and-feather disease, was detected in 6 healthy psittacines living in the same enclosure as those examined. This virus has a tropism for replicating cells, including growing feathers and immune cells, causing immunosuppression and allowing the development of secondary infections that can lead to death.³ Unfortunately, the presence of PsCV was not investigated in our 4 cases. In the lung of case 3, there were extensive areas of necrosis, some with degenerate heterophils clustered in the center or spread randomly. These areas also contained numerous basophilic fungal hyphae of ~ 3 μm diameter, with parallel walls, septation, and dichotomous branches, morphologically compatible with Aspergillus sp. A lung fragment was submitted to the Mycological Research Laboratory/UFSM where samples were cultured in a Sabouraud agar at 30°C for 5 d, confirming the growth of Aspergillus fumigatus. Impression smears from the crop of case 4 were Gram stained, and yeast structures compatible with Candida sp. were identified. Histologically in the crop, there was epithelial hyperplasia with hyperkeratosis, in the midst of which there were fungal yeast organisms (1–3 μm diameter) or basophilic pseudohyphae, morphologically compatible with Candida sp. Aspergillosis and candidiasis have also been described as secondary fungal infections in birds with respiratory disease caused by herpesviruses.²

The clinical and pathologic findings, supported by EM and molecular detection, indicate the involvement of PsHV-3 in the described outbreak. Our findings call attention to the circulation of this agent among Brazilian birds and reinforce the importance of adopting biosecurity measures to avoid the spread of pathogens.