Mortality due to polyomavirus infection in two nightjars (Caprimulgus europaeus).
Article Type: Report
Subject: Mortality (Netherlands)
Mortality (Germany)
Mortality (Portugal)
Mortality (Research)
Polyoma virus (Health aspects)
Polyoma virus (Research)
Authors: Arroube, Ana Sofia
Halami, Mohammad Yahya
Johne, Reimar
Dorrestein, Gerry M.
Pub Date: 06/01/2009
Publication: Name: Journal of Avian Medicine and Surgery Publisher: Association of Avian Veterinarians Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2009 Association of Avian Veterinarians ISSN: 1082-6742
Issue: Date: June, 2009 Source Volume: 23 Source Issue: 2
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: Netherlands; Germany; Portugal Geographic Code: 4EUNE Netherlands; 4EUGE Germany; 4EUPR Portugal
Accession Number: 252006953
Full Text: Abstract. Two nightjars (Caprimulgus europaeus) from a bird park in the Netherlands died suddenly, with no clinical signs, within 1 month of each other. The main pathologic findings at necropsy were splenomegaly and hepatic necrosis. On histologic examination, intranuclear viral inclusion bodies consistent with avian polyomavirus were observed in the liver, spleen, and kidneys. Polymerase chain reaction testing of samples from the liver, spleen, and kidneys detected avian polyomaviral DNA, and sequence analysis showed that the virus had a sequence homology of 99% to psittacine avian polyomavirus strains. To our knowledge, this is the first report of avian polyomavirus infection in the order Caprimulgiformes. Lovebirds (Agapornis species), which were housed near the nightjars, were considered as the possible source of infection.

Key words: polyomavirus, polymerase chain reaction, sequence analyses, avian, nightjars, Caprimulgus europaeus

Clinical Report

Two nightjars (Caprimulgus europaeus) were presented to the diagnostic pathology laboratory of the Dutch Research Institute for Avian and Exotic Animals in Veldhoven, The Netherlands, for necropsy. These nightjars originated from a bird park (Nederlands Opvang voor Papegaaien, Veldhoven, The Netherlands), where they were found dead in their cages. The first bird, a 2006 captive-bred male nightjar, died in March; it had been in the park for 4 months. The second nightjar, a 2005 captive-bred female, was found dead 1 month later; it had been in the park for 1.5 years. Neither bird showed any clinical symptoms. The birds were individually housed in indoor connecting cages (2 m high x 2 m long x 1.5 m wide). Many other birds were housed in the same room, but the only birds that had been moved into the room during the winter were a group of lovebirds (Agapornis species). These lovebirds were housed in a flight cage similar to the nightjar cages and were located 5 cages (7.5 m) from the nightjars. None of these lovebirds showed evidence of clinical disease or mortality.

On necropsy, both birds were in good body condition, which suggested an acute process, with no externally remarkable changes. In situ abundant coronary and coelomic fat was present. A superficial epicardial hemorrhage was present at the apex of the heart in both birds, and the female nightjar had mild hydropericardium. The lungs were minimally edematous. Approximately 1.5 ml of free clear fluid was present in the coelom of the female. The livers were the most affected organs and had a reddish-gray-orange color and a fine marble pattern. The liver of the male was very brittle, soft, but smooth; the liver of the female showed some swelling and was quite firm. The spleens were enlarged, dark red to red, and measured 10 and 13 mm long, respectively. No gross changes were present in other organs.

Results of a parasitologic examination of the intestines and direct microscopic examination by wet mount of the mucosa of the proventriculus for Macrorhabdus ornithogaster were negative. Cytologic examination (Hemacolor, Merck,

Darmstadt, Germany) of impression smears of the liver, spleen, lungs, and intestine was performed. The liver showed microvacuolization and some heterophils (without granules). The spleen showed a mixture of cells, including histiocytes, heterophils, monocytes, and granulocytes, with very fine basophilic granulation, but few lymphocytes and a minimal amount of erythropoesis. In the lungs, especially in the female, many heterophils, large monocytes, and also granulocytes with very fine basophilic granulation were seen. In the intestines of the male, many bacteria with different morphologies, including spore-forming bacteria and curly clostridial-like bacteria, were seen. In the female, only enterocytes were seen, with no bacteria. Because the organs of the male appeared pale at necropsy, a bone marrow sample was collected for cytologic examination. The bone marrow appeared hyperemic and showed production of heterophils but very little erythropoesis. Foamy mesothelial cells were present in the coelomic fluid of the female.

[FIGURE 1 OMITTED]

On histologic examination, the liver showed minor periportal lymphocytic infiltration and connecting centrolobular necrosis. In several liver cells, nuclei were enlarged, with marginating chromatin and with centrally containing basophilic inclusion bodies. In the spleen, extensive hemorrhages were present in the red pulp, and basophilic intranuclear inclusion bodies identical to those seen in the liver were present in the epithelial cells of the sheathed arteries (Fig 1). The kidneys showed extensive acute tubulonecrosis, and intranuclear inclusion bodies were present in the mesangium of the glomeruli. Therefore, the major histologic findings were hepatic necrosis, nephrosis, and splenitis, with intranuclear inclusion bodies.

Sections of the liver and spleen of both birds and of the kidney of the female were collected and stored at -20[degrees]C in 70% alcohol for viral testing. The liver samples fixed in alcohol were submitted for polymerase chain reaction (PCR) testing for avian polyomavirus (APV) (Gendika, Veendam, The Netherlands). Results showed that samples of both birds were positive for APV. The isolated DNA and the remaining tissue samples fixed in 70% alcohol of both birds were further analyzed.

The isolated DNA was amplified by 2 PCR protocols with APV-specific PCR primers (1) and nested consensus PCR primers. (2) The APV-specific PCR amplifies a PCR product with a length of 310 base pairs of the APV genome by using primers 5'-CAA GCA TAT GTC CCT TTA TCC C-3' and 5'-CTG TTT AAG GCC TTC CAA GAT G-3'. The nested consensus PCR amplifies a short region of the capsid proteinencoding region of all known polyomavirus genomes. Primers 5'-CCA GAC CCA ACT ARR AAT GAR AA-3' and 5'-AAC AAG AGA CAC AAA TNT TTC CNC C-3' were used in the first round of PCR, and primers 5'ATG AAA ATG GGG TTG GCC CNC TNT GYA ARG-3' and 5'-CCC TCA TAA ACC CGA ACY TCY TCH ACY TG-3' were used in the nested PCR amplifying a nested PCR product with a length of approximately 250 base pairs. By using either PCR protocol, the samples were positive. The APV-specific PCR products were sequenced, and a nucleotide sequence homology of 99% to the psittacine APV strain budgerigar fledgling disease virus-1 was determined by using a BLAST search of the GenBank database. DNA was again isolated from the tissues of the female (liver, spleen, and kidney) and male (liver and spleen), and was investigated by nested consensus PCR. Results from all organs were positive (Fig 2), and sequencing revealed a nucleotide sequence homology of 99% to the capsid protein encoding region of the psittacine APV strain budgerigar fledgling disease virus-1. The sequence data were submitted to GenBank (accession numbers EU315060 and EU315061). Blood samples that were collected from 4 lovebirds that were housed in the same room as the nightjars were fixed in 70% alcohol. All of these samples were negative by APV-specific PCR as well as by nested-consensus PCR testing. Other samples from the lovebirds, such as cloacal swabs or feathers, were not available for PCR testing.

Discussion

In this clinical report, we describe a fatal APV infection in 2 nightjars. The birds died within 1 month of each other, with no clinical signs. The main pathologic findings were hepatic necrosis and splenomegaly. On histologic examination, intranuclear inclusion bodies consistent with those of APV were observed in the liver, spleen, and kidneys. Avian polyomavirus DNA was detected by PCR testing of samples of liver and spleen of both birds, and kidneys of the female bird. Sequence analysis showed that the virus had a 99% sequence homology to psittacine APV strains.

[FIGURE 2 OMITTED]

Avian polyomavirus is the causative agent of budgerigar fledgling disease and was first described in young budgerigars (Melopsittacus undulatus).(3,4) A similar virus was also associated with high levels of morbidity and mortality in different genera of psittacine and nonpsittacine birds. (5 9) The nonpsittacine bird species includes those from the orders Galliformes, (10) Falconiformes, (1) and one report in a green aracari (Pteroglossus viridis, Piciformes). (11) In the Passeriformes, the infection was reported in Estrildidae, Fringillidae, Paridae, and in shamas (Copsychus malabaricus, Muscicapidae). (12-18) The nightjars we describe belong to the order Caprimulgiformes, and, to our knowledge there are no reports of polyomavirus infection affecting any representative of this order. The pathologic findings in these birds resembled those of polyomavirus infection in other birds. The diagnosis was based on histopathologic findings, PCR results, and sequencing of the PCR product.

The clinical and pathologic presentations in birds infected with APV depend on the species affected and the age and condition of the birds when exposure to the virus occurs. In budgerigars, as a general rule, disease is confined to nestlings, which develop a peracute or acute form, with high mortality. (3,4,9,19,20) The chicks can die suddenly, without symptoms or with signs like discolored skin, abdominal distension, ascites, subcutaneous hemorrhages, and with nervous signs, such as ataxia and tremors. (3,4,8,19,20) Survivors may exhibit symmetrical feather abnormalities. (8,19,21,22) Nestlings of nonbudgerigar psittacine birds with APV manifest the disease as an acute rapidly fatal disease, with no premonitory signs or with clinical signs similar to those described in budgerigar nestlings? (5,6,8,23) The disease in adult psittacine birds was also described. Although uncommon, the disease in these birds is characterized by the same signs and gross lesions as those seen in nestling parrots?

On pathologic examination, the liver and spleen are primarily affected. Hepatomegaly, yellow mottling of the liver, and splenomegaly may be seen. Swollen kidneys and congestion may also be present. (8,24) The gross pathologic findings in the nightjars were similar: hepatic necrosis, splenomegaly, and nephrosis in birds with an excellent body condition. The pallor of the male could not be explained by hemorrhages, which are usually seen in APV-infected birds. (8) In the bone marrow, erythropoesis was minimal, but, because no clinical data were available, the extent of the anemia could not be determined.

Liver necrosis is a common histologic finding in APV infections. In these 2 birds, a moderate, centrolobular necrosis was seen. The nuclei of infected cells were enlarged, had marginated chromatin, and centrally contained a finely granular basophilic-to-amphophilic inclusion, as is documented for other species. (8,24) Splenic and renal lesions are common in birds with APV, and this was also the finding in the nightjars. In the spleen, inclusion bodies are often abundant and are accompanied by necrosis of the perivascular histiocytes and other phagocytic cells. (24) In the nightjars, abundant intranuclear inclusion bodies were present, mainly in the sheathed arteries of the spleen and in the mesangium of the renal glomeruli. In other species, intranuclear inclusion bodies can occur in feather follicles, skin, esophagus, brain, and heart. (24)

In these 2 birds, infection with APV was confirmed by PCR testing. Sequencing of the PCR products resulted in a 99% homology on the nucleotide level with a psittacine APV strain. The high percentage of homology reflects the high genetic stability of APV and is in accordance with previous analyses of APV strains derived from different avian species. (1,10-25) However, psittacine APV is genetically different from other known polyomaviruses of birds, including goose hemorrhagic polyomavirus and the 2 polyomaviruses recently characterized as finch polyomavirus and crow polyomavirus. (25) Detection of psittacine APV in Caprimulgiformes confirms the broad host spectrum of this virus, which is in marked contrast to other known polyomaviruses. (26)

Disease typically follows 2 weeks after APV-free birds are introduced into a contaminated environment. (27) Asymptomatic birds indefinitely shed the virus at low concentrations, and the most likely means of entry of the virus is by inhalation. After infection, high concentrations of the virus are shed in the droppings. In budgerigars, the virus can also be shed in feather and skin dander, and may be present in oral secretions. Birds with inapparent infection and those that survive the clinical disease shed virus by these routes until at least 6 months of age. (22)

Because disease rarely occurs in adult birds, the nightjars were unlikely to have been primary carriers of the virus. The nightjars did not show clinical symptoms and had been in the park in the same housing for a long time: the male for 4 months and the female for 1.5 years. The only known change in their environment was the introduction of a group of lovebirds 1 month before the first nightjar died. Lovebirds are known to be susceptible to and can be subclinical carriers of polyomavirus. (22) Therefore, these lovebirds could have been the source of infection. Unfortunately, cloacal swab samples for direct testing of viral shedding by the lovebirds were not available at that time. Results of PCR testing of blood samples collected from the 4 lovebirds at a later time point were negative. However, viremia is short and may be intermittent during persistent APV infection of smaller psittacine birds, which may result in a negative PCR result. Although not definitively proven, in this case, it is likely that the lovebirds were subclinically infected (temporary) shedders and that they infected the nightjars. Because the male and female nightjars were healthy in the preceding 4 and 18 months, respectively, contamination of the environment before the lovebirds were introduced in the housing is not likely.

The findings in this report confirm that nightjars, which belong to the order Caprimulgiformes, not only can be infected by psittacine polyomavirus but can also develop a lethal disease. Therefore, the risks of housing nightjars together with species that are possible carriers of psittacine APV should be considered carefully. This report again demonstrates that APV is able to cross-infect birds that belong to totally different orders with a detrimental result.

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Ana Sofia Arroube, DVM, Mohammad Yahya Halami, Dr vet reed, Reimar Johne, Dr vet reed, PhD, and Gerry M. Dorrestein, DVM, PhD

From the Veterinary Faculty, Evora University, Portugal (Arroube); Institute for Virology, University of Leipzig, Faculty of Veterinary Medicine, Germany (Halami); Federal Institute for Risk Assessment, Berlin, Germany (Johne); and Diagnostic Pathology Laboratory, Dutch Research Institute for Avian and Exotic Animals, Veldhoven, The Netherlands (Dorrestein).
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