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Mixed infection with reovirus and Chlamydophila in a flock of budgerigars (Melopsittacus undulatus).

Abstract: Eleven budgerigars (Melopsittacus undulatus) from a zoological collection presented at necropsy with emaciation and splenomegaly or hepatomegaly or both. Polymerase chain reaction assays performed on liver and spleen samples were positive for Chlamydophila psittaci in 2 of 3 birds tested, and histologic findings in 2 additional birds were compatible with chlamydiosis. The aviary was subsequently closed to the public, and a 45-day treatment regimen with doxycycline in the seeds was initiated. No further deaths of birds with hepatomegaly or splenomegaly were observed after the first day of treatment. Further investigation of birds that died during the outbreak with emaciation and with hepatic and splenic enlargement revealed severe necrosis of the spleen and liver suggestive of reovirus infection, which was supported with polymerase chain reaction analysis from paraffin-embedded tissue. This reovirus was sequenced and had 100% homology with a reovirus previously identified in an African grey parrot (Psittacus erithacus). The outbreak did not affect cockatiels (Nymphicus hollandicus) and blue quail (Coturnix chinensis) kept in the same aviary. A group of budgerigars added to the collection soon before the opening of the aviary may have introduced reovirus and Chlamydophila into the collection.

Key words: reovirus, Chlamydophila psittaci, chlamydiosis, avian, budgerigar, Melopsittacus undulatus

Clinical Report

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.

Discussion

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]

[FIGURE 2 OMITTED]

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.

References

(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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Article Details
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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
Geographic Code:1USA
Date:Dec 1, 2010
Words:2451
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