Association of Avian Veterinarians Australasian Committee Ltd. Annual Conference 2016 pp 49-55 Adenovirus in Parrots: a Case Study Kathleen Fearnside,1 Shubhagata Das,2 Shane R. Raidal2

1 Normanhurst Veterinary Practice 2 School of Animal and Veterinary Sciences 31 Normanhurst Rd Faculty of Science Normanhurst, NSW 2076 Charles Sturt University, New South Wales [email protected] [email protected]

Introduction: cine adenovirus 1, 2 and 3 have been coined as a potential species based on limited adenovirus DNA A wide range of lesions have been associated with sequence data including the adenovirus hexon and adenovirus infection in birds including conjunctivi- DNA polymerase genes recovered from birds that tis, splenitis, hepatitis, enteritis and a wide variety have succumbed to fatal infection (Luschow et al., of other organs such as the central nervous system 2007; Katoh et al., 2009). However, only one full ge- through the damage caused to endothelium (Mori nome of a putative psittacine adenovirus, psittacine et al., 1989; Gomez Villamandos et al., 1995; Ritchie, adenovirus 3, is currently available in NCBI GenBank 1995; Schmidt et al., 2003; Katohi et al., 2010). Ad- (To et al., 2014). In this paper we present an investi- enoviruses are non-enveloped, double-stranded, gation of mortality in a mixed aviary flock of parrots linear, DNA viruses in the family Adenoviridae which and lorikeets that caused mortality only in red-bel- all replicate with high fidelity in the host nuclei of lied parrots (Poicephalus rufiventris). vertebrates (Ritchie, 1995). Adenovirus pathogens of commercial poultry and humans have been well Case Histories characterised in terms of their genetic diversity, pathogenicity and the diseases they cause. The Inter- Case 1: Red-bellied Prrot (Poicephalus rufiventris) national Committee on Taxonomy of Viruses (ICTV) - Male 1 recognises five genera in the family with the majority of avian adenoviruses so far characterised belonging In the first occurrence of disease there was sudden to the genus Aviadenovirus for which the type spe- death of a male red-bellied parrot (5th October cies is Fowl aviadenovirus A. This genus mostly af- 2013). The owner had purchased two pairs of appar- fects gallinaceous birds with diseases of commercial ently mature (4 years old) red-bellied parrots from significance in quail, poultry, ducks, geese, pheasants another breeder four months prior in June 2013. and turkeys. The predominance of avian adenovirus- There had been no problems prior to this. This bird es in gamefowl and waterfowl has hinted towards had been apparently normal the previous night. It an ancient host coevolution with the Galloanserae, looked ill that morning and died on the way to the however there is widespread serological evidence veterinary clinic. There was a very small amount of in many wild birds such as albatross (Padilla et al., blood soiling the vent and from this the owner was 2003), cormorants (Travis et al., 2006) and other avi- convinced to allow a post-mortem examination. Nec- an Neoavian lineages including pigeons, and parrots ropsy revealed significantly dilated and haemorrhag- points to a more widespread host diversity (Hess et ic intestines and mild pallor to liver. The presence of al., 1998a, 1998b; Mackie et al., 2003; Marlier et al., undeveloped testes and a well-developed bursa of 2006; Katohi et al., 2010) . Indeed avian hosts also Fabricius indicated that the bird was immature and dominate the related genus Siadenovirus, for which therefore unlikely to be four years of age. Samples of the type species is Frog siadenovirus A, but includes visceral organs were fixed in formalin. species from passerines, raptors and turkey (Katoh et al., 2009; Wellehan et al., 2009; Joseph et al., 2014). Histopathology of tissues demonstrated widespread Psittacine bird species are known to be susceptible severe congestion and necrosis with focal areas of to adenovirus infection but the ICTV has so far not enterocyte necrosis. In the liver and spleen there recognised any psittacine adenovirus species. Psitta- were multifocal areas of necrosis associated with www.aavac.com.au© 49 karyomegaly and basophilic intranuclear inclusions. The testicle was immature and appeared congested The kidney and testicles (immature) appeared con- but otherwise normal. The bursa of Fabricius was gested but otherwise normal. The bursa of Fabricius congested, oedematous and had moderate follicular was congested, oedematous and had moderate fol- lymphoid depletion. The proventriculus, ventriculus, licular lymphoid depletion. Accordingly a diagnosis lung and heart appeared mildly congested but nor- of acute viral hepatitis, splenitis and enteritis was mal. Blood, intestinal swabs and frozen spleen and made. Since no fresh samples were saved from liver samples tested PCR positive to psittacine ade- the bird for PCR testing the lesions were considered novirus 1 (Raue et al., 2005) and negative for avian most likely to be due to avian polyomavirus or psitta- polyomavirus and beak and feather disease virus us- cine adenovirus infection. The advice given at the ing established PCR protocols (Phalen et al., 1999; time was to provide careful monitoring of in-contact Ypelaar et al., 1999). PCR amplicon sequencing con- birds and to destroy contaminated equipment such firmed the detection of adenovirus DNA. as nest boxes and disinfect potentially contaminat- ed environment. The histopathological report noted Neither in-contact hen birds showed any sign of ill- that the immature testicle and presence of the bursa ness around the time that both cock birds died or of Fabricius indicated that this bird was less than 12 after the death of the second male bird. There was months of age. also no other unexplained deaths in nearby birds. Accordingly the owner was advised to wait at least Case 2: Red-bellied Parrot - Male 2 three months before sourcing and replacing the male birds. In February 2014 two new cock birds Some 13 days after the first male bird died the sec- were purchased and placed with the females. There ond male red-bellied parrot of the two pairs of birds was a small period where the older cock bird bullied became acutely lethargic. Akin to the first case this the hen when introduced but otherwise the health bird had been purchased as a three year old mature of the parrots was unremarkable until October 2014. bird four months prior along with a hen as a breed- ing pair. It was presented to the clinic on the 18th Case 3 Red-bellied Parrot - Male 3 October 2013 with acute onset of illness consisting of severe lethargy, mild dyspnoea and watery liquid One of the replacement male red-bellied parrots, on the cage floor. It was difficult to determine if the two years of age, was presented (7th October 2014) liquid was vomit or abnormal droppings. The own- with acute onset of severe lethargy. The owner had er reported that the bird was clinically normal four noticed that the bird had appeared unwell the pre- hours earlier. vious day where it had been sitting at the food dish and not eating with a progressive worsening of con- The bird was hospitalised and treated as for criti- dition. Clinically it was in reasonable body condition cal care. This included a heated hospital cage; fluid but was weak and lethargic with a partially distended therapy (s/c Hartmann’s solution with 5% glucose); crop containing fluid. Non-invasive faecal and a crop an oxygen cage PRN; amoxicillin/clavulanic acid 125 wash wet-preparation microscopy were unremark- mg/kg s/c SID; and Enrofloxacin 30 mg/kg SID. able. The bird was hospitalised and treated aggres- sively as for critical care for bird 2 (above). However, The bird continued to deteriorate and died the fol- the bird continued to deteriorate and passed very lowing day, 22 hours after presenting. Necropsy -ex bloody droppings a few hours later with haemor- amination revealed a mottled liver and again, unde- rhagic diarrhoea, and died overnight. Post-mortem veloped testes indicating a sexually immature bird. examination revealed significantly dilated and haem- Histopathological examination confirmed a diagno- orrhagic intestines with bloody fluid contents in the sis of acute necrotising viral hepatitis and enteritis. cloaca. The liver was pale and the spleen was grossly There was moderate autolysis which interfered with normal size. There were intramuscular haemorrhag- histological interpretation. Nevertheless, throughout es in the wall of the proventriculus. Small undevel- the intestine there was widespread congestion and oped testes were present. The post-mortem findings haemorrhage and focal areas of enterocyte necrosis. were very similar to those seen one year earlier in In the liver there were multifocal areas of necrosis the first two cases. Subsequent histopathological associated with karyomegaly and basophilic intranu- examination demonstrated widespread severe con- clear inclusions. The kidney was moderately congest- gestion throughout the intestine with massive hae- ed and scattered tubules had intraluminal uric acid morrhage into the lumen and widespread necrosis tophi likely associated with terminal dehydration. of enterocytes with occasional basophilic intranucle- www.aavac.com.au© 50 ar inclusions and karyomegaly, mostly within endo- and two days after the first cases were seen and co- thelial cells of the lamina propria. In the liver and incided with the first period of hot weather of the spleen there were multifocal areas of necrosis asso- 2014 breeding season. The clinical presentation ciated with karyomegaly and basophilic intranuclear and histopathology were similar and only the male inclusions, mainly in the liver. The lung, heart, brain, red-bellied parrots were affected so it seemed plau- kidney and appeared congested but otherwise nor- sible that a carrier status may have been present in mal. Akin to the previous cases a diagnosis of acute the flock, perhaps one of the female birds oroth- necrotising viral hepatitis, splenitis and haemorrhag- er species held at the property. The two banks of ic enteritis with intralesional viral inclusions consis- suspended wire aviaries also housed Senegal parrots tent with acute adenovirus infection was concluded. and purple-crowned lorikeets.

Case 4. Red-bellied Parrot - Male 4 Attempts to identify carrier birds In an attempt to identify adenovirus carrier birds, The second replacement male Red-bellied Parrot blood and cloacal samples were collected from the was presented to the clinic the following day, after in-contact female red-bellied parrots and nearby the overnight death of Bird 3 (8th October 2014). birds on the 30th October 2014. There had been This bird was reportedly four years of age and the no further losses at this time. An aviary visit was ar- owner reported that, whilst it had behaved nor- ranged to assess the management options for the mally during the previous day, it had deteriorated different aviary flock subsets. There were several in behaviour alongside with Case 3. Clinically it was banks of aviaries housed on a standard house block, in moderate body condition and mildly lethargic. It some with a single species and others with a mixed had an empty crop and the droppings were grossly open aviary flock. Other species of birds held by normal. Non-invasive faecal microscopy and a crop the collector included two pairs of Senegal parrots, wash were unremarkable. The bird was hospitalised purple crowned lorikeets, elegant parrots, scarlet and treated aggressively as for critical care for bird parrots, doves, finches and lovebirds. Local wildlife 2 (above). The bird ate well that night but died the including wild birds that visited the roof of aviaries following morning. The droppings passed overnight were also abundant and there were several rainbow were grossly normal. Post-mortem examination re- lorikeets and sulphur crested cockatoos present on vealed significantly watery fluid filled intestines; the day of the site visit. Disinfection of aviary walls food in the crop and some vomit/regurgitation in and substrates was considered not feasible due to the oral cavity. The liver was grossly normal and the difficulty of access below the suspended wire aviar- spleen mildly enlarged. The testes were moderately ies as well as the use of dirt and sand as a floor sub- hypertrophic and sexually mature. Cytological ex- strate in free-flight aviaries. Inter-flock quarantine amination of liver impression smears demonstrat- was considered possible but difficult with relation to ed many hepatocytes with massively enlarged and the arrangement of aviaries and stock density. strongly staining bluish-purple intranuclear inclu- sions consistent with an acute DNA viral infection As shown in Figure 1, there were two banks of sus- such as psittacine adenovirusor avian polyomavirus. pended aviaries opposite each other separated by a Subsequent histopathological examination revealed narrow aisle in which the male red-bellied parrots severe congestion throughout the small and large in- which had died had been housed. These are situat- testine including the cloaca where there was focal to ed on the southern corner of the property. The two widespread areas of epithelial necrosis. In the liver pairs of red-bellied parrots and a pair of Senegal par- there were multifocal areas of necrosis associated rots were housed separately in the south-western with marked karyomegaly and basophilic intranucle- back (A-C). The north-eastern side of these aviaries ar inclusions. The lung was markedly congested. The consisted of four smaller suspended aviaries (D-G), brain, skin, kidney and testicle (immature) appeared housing purple crowned lorikeets. During the site congested but otherwise normal. PCR testing was visit samples were collected consisting of blood and positive for psittacine adenovirus 1 and negative for cloacal swabs from both widowed red-bellied parrot avian polyomavirus and beak and feather disease vi- hens; blood and cloacal swabs from the pair of Sene- rus. gal parrots adjacent to the red-bellied parrots; blood and cloacal swabs as well as nest material from a pair In both Cases 3 and 4 the pathological findings and of purple crown lorikeets housed in an aviary directly test results were very similar to the first two cases opposite to the affected red-bellied parrots. Cloa- seen the previous year. Cases 3 and 4 died one year cal swabs and nesting material were collected from www.aavac.com.au© 51 Figure 1. Layout of facility showing location of suspended wire aviaries (A-G) in relation to a larger open flighted aviary with earthen floor (H).

all other nearby purple crown lorikeets (nine birds tion. At the owners request the mate of this bird and and three nests). All nest material samples were its single offspring were euthanased for diagnostic PCR negative for adenovirus by PCR. Three purple testing. These were PCR negative for adenovirus and crowned lorikeets were positive (Table 1). avian polyomavirus.

No deaths occurred in the aviary flocks for five Repeat sampling and testing were organised with clo- months, when on the 20th ch 2015 the owner re- acal swab samples collected from five clinically nor- ported that the female purple crowned lorikeet (ID mal breeding purple crowned lorikeets in November 10-36 gold) that was housed in aviary E, directly op- 2014 which identified two birds as PCR positive for posite the affected red-bellied parrots had died a lit- adenovirus and were assumed to be shedding the vi- tle over one week earlier and was lost to investiga- rus. Birds in this colony, and not necessarily the pair www.aavac.com.au© 52 Table 1. Adenovirus PCR Testing (30th October 2014)

Aviary Bird ID and sex (male/unknown/female) Sample Adenovirus

G Purple crowned lorikeet (u) Swab Positive G Purple crowned lorikeet Captn 78 split (u) Swab Positive G Purple crowned lorikeet Kirk 61 split (u) Swab Negative G Purple crowned lorikeet Kirk 63 split (u) Swab Positive G Purple crowned lorikeet Yellow Kirk split (u) Swab Negative G Purple crowned lorikeet CAPTN 77 split (u) Swab Negative F Purple crowned lorikeet KIRK 55 (m) Swab Negative F Purple crowned lorikeet 09-02 (f) Swab Negative E Purple crowned lorikeet 08-01 black (m) Blood Negative E Purple crowned lorikeet 08-01 black (m) Swab Negative E Purple crowned lorikeet 10-36 gold (f) Blood Negative E Purple crowned lorikeet 10-36 gold (f) Swab Negative D Purple crowned lorikeet CAPTN 08 + 10-49 (f) Swab Negative D Purple crowned lorikeet 10-06 (u) Swab Negative C Red-bellied parrot Matt-10-008 (f) Blood Negative C Red-bellied parrot Matt-10-008 (f) Swab Negative A Red-bellied parrot Matt-10-005 (f) Blood Negative A Red-bellied parrot Matt-10-005 (f) Swab Negative B Senegal parrot (f) Blood Negative B Senegal parrot (f) Swab Negative B Senegal parrot (m) Blood Negative B Senegal parrot (m) Swab Negative that tested positive, had bred successfully without of the disinfectant F10 1:250 dilution over 2 weeks. nestling or fledgling mortality. On the 25th July 2015 The owner was also advised to wait at least two this colony was removed and relocated to another months before introducing new male red-bellied premises in an effort by the owner to de-populate parrots which were being held in pre-sale quaran- and remove potential adenovirus carriers. The own- tine at the seller’s property. In follow-up communi- er was advised to remove all other purple crowned cations with the owner on the 9th November 2015 lorikeets from that bank of aviaries and house them two new male red-bellied parrots had settled in well much further away. The owner was advised to thor- with no deaths or evidence of illness in any of the oughly clean the entire bank of aviaries, including red-bellied and Senegal parrots, even with six weeks those with the red-bellied parrot hens and pair of of very warm weather. Senegal parrots, followed by at least two treatments www.aavac.com.au© 53 Repeat testing of suspect adenovirus carrier purple grey parrots (Droual et al., 1995). Group I avian ade- crowned lorikeets novirus infection is often subclinical with antibodies At the time they were relocated from the owner’s frequently present in healthy chickens, turkeys and property the purple crowned lorikeet colony with other avian species (McFerran et al., 1977, 2000; the suspect adenovirus carriers comprised two pairs Mackie et al., 2003). of adult purple crowned lorikeets and their eight off- spring, including a recently hatched nestling. They The significance of infection by a novel adenovirus were clinically normal. On the 20th March 2016 they most closely related to Fowl adenovirus C in psitta- were retested PCR negative for adenovirus. cine birds remains unclear. However, it is evident that psittacine birds should be considered suscepti- Adenovirus Genome sequence analysis ble to a wide range of avian adenovirus species from Liver tissue from three affected red-bellied- par different host sources (Gomez Villamandos et al., rots was used to extract and analyse viral DNA us- 1995; Ritchie, 1995; Schmidt et al., 2003; Katohi et ing Next-Generation sequencing. This produced full al., 2010; Joseph et al., 2014). Adenovirus infections length virus genomes with near identical similarity have been found in apparently healthy birds and to one another with a genome architecture most carrier states may exist in certain species or within closely aligned and 51.8% similar to Fowl adenovi- individually infected birds (Wellehan et al., 2005; Lu- rus C and only 29.5% similar to psittacine adenovi- schow et al., 2007) and it has been suggested that rus 3. The hexon gene had 73-82% similarity with stress may precipitate the overt viral replication in fowl, psittacine and pigeon adenovirus hexon genes the pathogenesis of necrotising lesions (Droual et whereas the DNA polymerase gene had 62% similar- al., 1995). ity to Fowl aviadenovirus C DNA polymerase. References Discussion Dean J, Latimer KS, Oaks JL, Wuenschmann A. 2006. In this paper we present an investigation of mortal- Falcon adenovirus infection in breeding Taita falcons ity caused by adenovirus infection in a mixed aviary (Falco fasciinucha). Journal of Veterinary Diagnostic flock of parrots and lorikeets that caused mortality Investigation. 18, 282-286. only in red-bellied parrots (Poicephalus rufiventris). A recent survey of captive parrot populations in Vic- Droual R, Woolcock PR, Nordhausen RW, Fitzgerald toria found a low prevalence of adenovirus infection SD. 1995. 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