Mixed infection with reovirus and Chlamydophila in a flock of budgerigars (Melopsittacus undulatus).
Key words: reovirus, Chlamydophila psittaci, chlamydiosis, avian, budgerigar, Melopsittacus undulatus
On July 5, 2007, a new outdoor walk-through aviary was opened at the Henry Doorly Zoo in Omaha (Nebraska, USA). The aviary was started with 425 budgerigars (Melopsittacus undulatus), 13 cockatiels (Nyrnphicus hollandicus), 10 blue quail (Coturnix chinensis), and 3 crested pigeons (Ocyphaps lophotes). Most of the budgerigars (325) had been maintained at the zoo since their purchase from a commercial farm in May 2006, but another 100 budgerigars were purchased in June 2007 and were added to the aviary after a 23-day period of quarantine. In addition, 200 budgerigars from the same commercial farm were purchased and added to the collection on July 24, 2007, without previous quarantine. Mortality started soon after the aviary was opened to the public. Most birds presented for necropsy had lesions attributed to predation or human-related trauma; however, several budgerigars had marked cachexia (Fig la), hepatomegaly (Fig 1b) and splenomegaly (Fig 1c and d). Affected birds had a mean weight of 27 g (n = 9), which was considerably lower than the mean weight of birds that died from predation or human-related trauma (mean, 35 g; n = 13). Livers were markedly enlarged and irregularly mottled tan-brown, and splenic enlargement up to 1 cm in diameter was seen in most birds. Other less common findings were conjunctivitis (Fig 1e), airsacculitis, pericloacal feathers stained with green feces (Fig 1f), and uric acid deposits in kidneys (Table 1). Initial studies revealed positive results of Chlamydophila polymerase chain reaction (PCR) assay on 3 of 4 swabs from spleen and liver samples (Table 1). Aerobic culture results from the liver and spleen of 1 budgerigar (bird 3) were negative. Initial histologic lesions reported were hepatocellular necrosis with histiocytic infiltrates; necrosis, histiocytosis, and hyperplasia of the spleen; and fibrinonecrotic airsacculitis. Because of laboratory technical problems at that time, Chlamydophila immunohistochemistry could not be performed. A presumptive diagnosis of chlamydiosis was made, and the aviary was closed to the public on August 1, 2007. The remaining flock was treated with doxycycline (300 mg/kg seed for 45 days). (1) The initiation of therapy was associated with cessation in the mortality associated with lesions of hepatosplenomegaly and emaciation. The same day that the treatment was started, 5 budgerigars with cachexia and an inability to fly were removed from the aviary, put in an indoor cage, and treated with the same protocol. All 5 birds recovered after treatment; however, these budgerigars were never tested for any infectious disease. Chlamydophila PCR was performed in fecal samples from 5 budgerigars from the main aviary after treatment, all with negative results. Overall, 11 affected budgerigars died between July 5 and August 1, 2007, which accounted for 13.5% of the total mortality
for the 4 months after the aviary was opened. No further mortality related to cachexia and hepatosplenomegaly occurred in the flock during the subsequent 24 months.
Samples from some of the affected budgerigars that died were subsequently reviewed at another laboratory. Histologic lesions reported included histiocytosis, congestion, hemorrhage, and lymphoplasmacytic inflammation of the spleen and extensive extramedullary hematopoiesis, lymphoplasmacytic inflammation, congestion, hemorrhage, and necrosis of the liver (Fig 2). Hepatic and splenic lesions were compatible with a viral agent, typically reovirus. In some cases, air sacs were thickened and fibrinonecrotic. Avian gastric yeast (Macrorhabdus ornithogaster) and adenovirus nephropathy were also found, but they also were present in budgerigars that died from trauma. Gimenez stains for the demonstration of chlamydial bodies were positive in 3 of 6 birds, although the number of infected cells was considered very low in comparison with birds that typically die from chlamydiosis. Formalinfixed paraffin-embedded samples from 6 budgerigars and frozen samples from another budgerigar were submitted to the University of Florida for heminested consensus reovirus PCR assay and sequencing. The protocol was adapted from a published protocol, (2) but, to increase chances of success with probable shorter fragments of RNA present in formalin-fixed paraffin-embedded tissues, a shorter target was selected. The first round used forward primer 2090F (5'-GGBTC MACNGCYACYTCBACYGAGCA-3') and reverse primer 2334R (5'-CDATGTCRTAHWYC CANCCRAA-3'), which was the second round of the original protocol. The second round used 2090F and a different reverse primer, 2200R (5'CCRTCRTCWCCYTGRCAKACRTARTT-3'). Reaction compositions and conditions were otherwise identical to those previously reported. Only 1 of the formalin-fixed paraffin-embedded samples produced an amplicon of the correct size consistent with reovirus (Table 1). Direct sequencing confirmed 100% homology with a reovirus previously identified in an African grey parrot (Psittacus erithacus) (Genbank accession number EU309695). (2) The remaining samples were negative for reovirus by PCR, including the frozen sample (which was tested for Chlamydophila psittaci by PCR with positive results). Electron microscopy was performed on one sample (bird 11) and no particles compatible with reovirus were found.
Avian orthoreoviruses are nonenveloped, double-stranded RNA viruses that belong to the genus Orthoreovirus in the family Reoviridae. (2) Although they are not always pathogenic, avian reoviruses may cause serious disease in birds, especially in poultry. (3) Reovirus infection has been reported in several psittacine bird species, with most cases affecting African grey parrots, (4-7) Australian king parrots (Alisterus scapularis), (7) and budgerigars. (3,8,9) Clinical signs reported with reovirus infection in psittacine birds are variable and include ruffled feathers, anorexia, lethargy, nasal discharge, respiratory signs, diarrhea, and sudden death. (3,5,7) Gross lesions include atrophy of pectoral muscles, lack of fat, enlarged liver, enlarged spleen, pale small foci in liver and spleen, swollen kidneys, ascites, airsacculitis, subcutaneous hemorrhages, lung congestion, and pneumonia. (3,5-7) In budgerigars, gross lesions have included poor body condition and hepatosplenomegaly, with airsacculitis, congestion of the lungs, pale kidneys, and staining of the vent with feces occurring less frequently (3); these gross findings are very similar to the ones we report. Histologic lesions in different species of psittacine birds infected with reovirus include variable degrees of necrosis, lymphoplasmacytic infiltrates, hemosiderosis, hemorrhage, and fibrin deposition in liver and spleen, necrosis and tubular degeneration in kidneys, enteritis, degeneration of the myocardium, lymphoid depletion in the bursa of Fabricius, and inconsistent necrosis and lymphoplasmacytic infiltrates in other organs, such as pancreas, lung, bone marrow, and testes. (3,5-7) Shipment and introduction of new birds are usually associated with the initiation of an outbreak of avian reovirus, (3,6,7) and both situations occurred in the present report. Therefore, the budgerigars added to the collection soon before the opening of the aviary may have introduced reovirus and Chlarnydophila into this collection.
[FIGURE 1 OMITTED]
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Reovirus infections in birds are commonly associated with other pathogens. (4,5) Experimental infections in African grey parrots could not reproduce all the clinical signs and gross lesions seen in the birds from which the virus had been recovered; this suggests that some lesions found in birds with natural reovirus infections are the result of concurrent conditions and not the results of the virus itself. (5) As an example, gout in the budgerigars we describe was attributed to renal adenovirus infection and dehydration, and conjunctivitis and airsacculitis may have been caused by the Chlamydophila infection. Immunosuppression induced by avian reoviruses can predispose the host to other pathogens and account for the diversity of syndromes associated with reoviruses. (6,10,11) In this report, concurrent infection with reovirus and Chlarnydophila were thought to be responsible for the mortality observed. Chlamydophila was detected by PCR and Gimenez stain of the splenic samples, but the extent of hepatic necrosis and the general absence of Chlarnydophila in necrotic lesions in the spleen and liver suggests that other agents may have contributed to the lesions, possibly reovirus. The strong association between the start of the treatment for avian chlamydiosis and the stop of the mortality suggests that other pathogens (in this case, Chlarnydophila) need to be present for this reovirus to cause mortality in budgerigars. Therefore, we hypothesize that the African grey parrot reovirus (2) is not highly pathogenic for budgerigars.
Avian gastric yeast and adenovirus infections may have been facilitated by reovirus-associated immunosuppression or may have been coincidental, because those 2 infectious agents were also found in budgerigars that died from other causes. Although reovirus infection in budgerigars has been associated with high mortality in flocks in the United Kingdom, (8,9) we did not observe high mortality because of reovirus in our flock of budgerigars; this suggests that either budgerigars in the United Kingdom were infected with a reovirus different than the African grey parrot reovirus or that the therapeutic protocols applied were not effective to control concurrent infections. We recommend that flocks that undergo mortality associated with reovirus should be treated with supportive care, antibiotics, and antifungals, and efforts should be made to try to characterize concurrent infections.
The recovery rate from paraffin samples is 20% or less for some DNA viruses such as adenoviruses (12); this number should be lower for RNA viruses such as reoviruses, and this may account for the negative reovirus PCR results for 5 of the 6 birds tested. Although there was a strong association between the start of treatment and the stop of mortality, this association could have been coincidental or caused by the immunomodulatory, rather than the antimicrobial, effects of doxycycline. (13,14) In addition, the aviary was closed to the public during treatment, which could have reduced the stress levels in the budgerigars and improved the outcome of treatment. Also, undetected agents other than Chlamydophila and reovirus possibly contributed to lesion development and severity in these birds.
(1.) Flammer K, Trogdon MM, Papich M. Assessment of plasma concentrations of doxycycline in budgerigars fed medicated seed or water. J Am Vet Med Assoc. 2003;223:993-998.
(2.) Wellehan JFX, Childress AL, Marschang RE, et al. Consensus nested PCR amplification and sequencing of diverse reptilian, avian, and mammalian orthoreoviruses. Vet Microbiol. 2009;133:3442.
(3.) van den Brand JMA, Manvell R, Paul G, et al. Reovirus infections associated with high mortality in psittaciformes in The Netherlands. Avian Pathol. 2007;36:293-299.
(4.) Wilson RB, Holscher M, Hodges JR, Thomas S. Necrotizing hepatitis associated with a reo-like virus infection in a parrot. Avian Dis. 1985;29:568-571.
(5.) Graham DL. Characterization of a reo-like virus and its isolation from and pathogenicity for parrots. Avian Dis. 1987;31:411-419.
(6.) Sanchez-Cordon PJ, Hervas J, Chacon de Lara F, et al. Reovirus infection in psittacine birds (Psittacus erithacus): morphologic and immunohistochemical study. Avian Dis. 2002;46:485-492.
(7.) Conzo G, Magnino S, Sironi G, et al. Reovirus infection in two species of Psittaciformes recently imported into Italy. Avian Pathol. 2001;30:43-47.
(8.) Pennycott T. Mortality in budgerigars in Scotland: pathological findings. Vet Rec. 2004;154:538-539.
(9.) Manvell R, Gough D, Major N, Fouchier RA. Mortality in budgerigars associated with a reovirus-like agent. Vet Rec. 2004;154:539-540.
(10.) Ni Y, Kemp MC. A comparative study of avian reovirus pathogenicity: virus spread and replication and induction of lesions. Avian Dis. 1995;39:554-566.
(11.) Montgomery RD, Villegas P, Dawe DL, Brown J. Effect of avian reoviruses on lymphoid organ weights and antibody response in chickens. Avian Dis. 1985;29:552-560.
(12.) Garner MM, Wellehan JFX, Pearson M. Pathology and molecular characterization of two novel atadenoviruses in colubrid snakes. J Herpetol Med Surg. 2008;18:86-94.
(13.) Brown DL, Desai KK, Vakili BA, et al. Clinical and biochemical results of the metalloproteinase inhibition with subantimicrobial dose of doxycycline to prevent acute coronary syndromes (MIDAS) pilot trial. Arterioscler Thrornb Vase Biol. 2004;24:733-738.
(14.) Woo PCY, Tsoi H-W, Wong L-P, et al. Antibiotics modulate vaccine-induced humoral immune response. Clin Diagn Lab Immunol. 1999;6:832-837.
David Perpinan, LV, MSc, Dipl ECZM, Michael M. Garner, DVM, Dipl ACVP, James F. X. Wellehan, DVM, MS, Dipl ACZM, Dipl ACVM, and Douglas L. Armstrong, DVM
From the Center for Conservation and Research, Henry Doorly Zoo, 3701 South 10th St, Omaha, NE 68107, USA (Perpinan, Armstrong); Northwest ZooPath, 654 W Main St, Monroe, WA 98272, USA (Garner): and the Zoological Medicine Service, Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, PO Box 100126, Gainesville, FL 32610, USA (Wellehan). Present address (Perpinan): Balmes 17, Badalona 08918, Barcelona, Spain.
Table 1. Clinicopathologic findings in budgerigars that died with cachexia, hepatomegaly, and-or splenomegaly during a 26-day period after introduction of a new group of budgerigars to the flock. Bird Weight, Gross lesions no. g 1 23 Cachexia, hepatosplenomegaly 2 27 Cachexia, hepatosplenomegaly, uric acid deposits in the kidneys 3 29 Cachexia, hepatosplenomegaly 4 NR Cachexia, hepatosplenomegaly 5 34 Splenomegaly (trauma-related death) 6 26 Cachexia, hepatosplenomegaly 7 25 Cachexia, hepatosplenomegaly, pericloacal feathers stained with green feces 8 NR Cachexia, hepatosplenomegaly 9 26 Cachexia, hepatosplenomegaly 10 26 Splenomegaly (trauma-related death) 11 23 Cachexia, hepatosplenomegaly, conjunctivitis, airsacculitis, pericloacal feathers stained with green feces Bird Histologic lesions Gimenez Chlamydophila Reovirus no. stain PCR PCR 1 NP NP NP NP 2 Necrotizing hepatitis; - NP - histiocytic splenitis; interstitial pneumonia; renal tubular necrosis and interstitial nephritis; chronic enteritis; myocardial necrosis; atrophy of fat and pancreas 3 Splenitis; hepatitis; Mildly + - - acute interstitial pneumonia; ventricular ulceration; chronic coelomitis; myocardial necrosis 4 Necrotizing hepatitis; - + - histiocytic splenitis 5 NP NP NP NP 6 Necrotizing hepatitis Mildly + + - and splenitis, chronic interstitial nephritis, proventriculitis with Macrorhabdus 7 Necrotizing hepatitis; - NP - congestion, hemorrhage, necrosis and fibrin deposit in spleen; congestion, hemorrhage, edema, and atelectasis in lungs; mild rhabdomyolysis; lymphoplasmacytic enteritis; lymphoplasmacytic tubulointerstitial nephritis 8 NP NP NP NP 9 NP NP NP NP 10 NP NP NP NP 11 Hepatic necrosis and Mildly + + + hepatitis; plasmacytic and/or histiocytic splenitis; atrophy of fat Abbreviations: PCR indicates polymerase chain reaction; NP, not performed; NR, not recorded.
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|Title Annotation:||Clinical Reports|
|Author:||Perpinan, David; Garner, Michael M.; Wellehan, James F.X.; Armstrong, Douglas L.|
|Publication:||Journal of Avian Medicine and Surgery|
|Article Type:||Clinical report|
|Date:||Dec 1, 2010|
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