Aspergillosis in a Blue-Fronted Amazon Parrot (Amazona aestiva): A Study Case

Julie Eifert, DVM; Heather L. Tarpley, DVM; Kenneth S. Latimer, DVM, PhD.

Class of 2005 (Eifert) and Department of Pathology (Tarpley, Latimer) College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7388

Signalment – Avian, Blue-Fronted Amazon Parrot, unknown sex, 3-year-old.

Presenting problem – Four week history of weight loss, one to two week history of decreased drinking, and decreased appetite.

History – The patient was taken to the referring veterinarian on 6/23/04, where a one-week course of enrofloxacin was prescribed but did not improve the bird’s condition. The bird was then given doxycycline and rehydrated with Pedialyte® for approximately 4 days until it was presented to the University of Georgia Veterinary Medical Teaching Hospital. Five other psittaciform birds of various breeds were kept in the same room as this bird but were housed in separate cages. The bird occasionally had contact with a Severe Macaw for short periods of time.

Physical Examination – The bird was bright and alert upon presentation. Neither crackles nor wheezes of the lungs were heard upon auscultation. There was no evidence of ocular or auricular discharge or other abnormalities. Vital signs included a pulse of 360 beats per minute and a respiratory rate of 20 breaths per minute. The bird had a body condition score of 1.5 / 5. All other physical examination findings were unremarkable.

Laboratory data -

*Oral itraconazole (initially at 11 mg/kg BID, later decreased to SID) was instituted on 7/2/04 for 4-8 weeks, along with nebulization therapy with amphotericin B (50 mg reconstituted with 10 ml sterile water, further diluted in 6 ml sterile water and infused over 15-25 minutes) for 5 days BID starting on 7/2/04.

Radiographic findings – Whole-body ventrodorsal radiographs displayed ill-defined soft tissue density in the left caudoventral coelom.

Gastro-intestinal barium contrast study findings – Normal emptying of gastrointestinal tract; no remarkable findings.

Endoscopy findings – Upon entering the left thoracic air sac via coeleoscopy, a small focal mass of granulomatous material was observed caudally. Upon entry into the abdominal air sac, severe infiltration of white, coarse, granulomatous material was observed, which was strongly suggestive of a fungal infection (Fig. 1 and 2).

Figure 1. Cytologic appearance of fungal hyphae with parallel walls, septa, and branching consistent with Aspergillus sp. (respiratory aspergillosis, Wright stain).	Figure 2. Cytologic appearance of macroconidium (left side of image) covered with slightly refractile fungal spores (respiratory aspergillosis, Wright stain).

Fungal culture of direct swab of the abdominal air sac – Light growth of Aspergillus sp. (4 weeks post culture inoculation)

1. Aspergillosis with profound leukocytosis characterized by a mature heterophilia and a severe lymphopenia. Leukocytosis is associated with inflammatory diseases, such as bacterial and fungal infections (including mycobacteriosis and chlamydiosis) and trauma. Other less likely causes include hemorrhage into the coelomic cavity, toxicities, neoplasia, and leukemias. Furthermore, leukocytosis with a heterophilia is often observed with infectious diseases caused by bacteria (including Chlamydophila sp.), fungi, and parasites. An extremely elevated heterophil count is common in aspergillosis, and usually ranges from 15,000 to 40,000 cells /µl (88,000 cells /µl in present case). Endogenous release of corticosterone in birds experiencing inflammation or infection can often cause lymphopenia due to the temporary redistribution of recirculating lymphocytes, as seen in this patient.

2. Late-stage monocytosis with eosinophilia. Monocytosis is often indicative of granulomatous disease or extensive necrosis in which a large amount of debris is being phagocytized, such as this case of severe infiltrative aspergillosis. The function of avian eosinophils is unclear. They are thought to modulate delayed type-IV hypersensitivity reactions. They are also thought to participate in inflammatory responses, phagocytosis, as well as bactericidal and parasiticidal activities. Severe Aspergillus sp. infection is likely to cause a moderate eosinophilia.

3. Severe hypoalbuminemia. Albumin functions as an osmotic pressure regulator and a transport protein. It is the largest protein fraction in serum. Hypoalbuminemia can occur in chronic infectious diseases, including (but not limited to) aspergillosis, chlamydiosis, and tuberculosis. In these instances, total protein is often within normal limits with a decreased A/G ratio. An inflammatory process will also result in a decrease in albumin concentration, and usually an increase in total protein concentration due to the increase in alpha-, beta-, and gamma-globulins. Occasionally, a decrease in albumin concentration can be seen with chronic liver disease, renal disease, parasitism, gastrointestinal disease, and over-hydration, all of which are much less likely causes in this patient.

4. Moderate hypokalemia. A decrease in serum potassium concentration may result from electrolyte loss through diarrhea (not present in this case) or during states of metabolic alkalosis where potassium ions are substituted for intracellular hydrogen ions. Malnutrition can also cause hypokalemia and is probably a contributing factor in this patient related to inappetance.

5. Metabolic alkalosis. An increase in bicarbonate concentration indicates a metabolic alkalosis. Loss of HCl is almost always the cause of metabolic alkalosis, and may be accounted for by secondary gastrointestinal stasis in this bird.

6. Elevated creatine kinase activity. The primary sources of creatine kinase are skeletal muscle, cardiac muscle, and nervous tissue. Elevation of the activity of this enzyme is usually a result of disruptions of any of these tissues. Transport and over-activity due to struggling in a stressful setting is enough to cause the increase in creatine kinase activity seen with this case. Furthermore, improper venipuncture technique with penetration of skeletal muscle also will result in increased creatine kinase activity.

7. Decreased lactate dehydrogenase (LDH) activity. LDH is an enzyme found in muscle, liver, kidney, and erythrocytes. Because there are many sources for the enzyme, many consider it to be an unreliable detector of disease, especially when enzymatic activity is decreased. It should be noted, however, that decreased LDH activity can be seen in end-stage liver disease, although this is unlikely in a bird with this presentation. Additionally, reference intervals for LDH are for serum samples. If samples are submitted in sodium-heparin tubes, results may appear to be out of the reference interval.

8. Mildly increased amylase activity. Amylase is produced by the pancreas, liver, and small intestine. Only elevations of two to three times above the reference range are significant and usually indicate acute pancreatitis or enteritis. This finding, therefore, is not significant with this bird’s presentation.

Discussion – Aspergillus sp. is a ubiquitous fungus that is found in the natural environment. Furthermore, it is the most common fungal agent involving respiratory disease in birds. Aspergillus sp. often causes disease in pet psittaciform birds, especially in Amazon parrots and African grey parrots. The causative agent is usually Aspergillus fumigatus, although Aspergillus flavus and Aspergillus niger infections can also occur.

Aspergillus sp. is transmitted by spores from environments favorable for fungal growth. Poor husbandry (i.e., inadequate ventilation, moldy food, etc.) can predispose birds to this disease. Aspergillosis can also be a secondary infection in an already stressed, debilitated, or immunosuppressed bird. This bird had a leg injury about one month before the initial visit to the referring veterinarian. The leg injury was not treated and healed on its own, but it could have caused enough stress or debilitation to immunocompromise the bird.

Aspergillus sp. can cause disseminated or locally infiltrative disease; the most common presentation is diffuse lower respiratory tract disease with syringeal and focal CNS (brain) granulomas also being somewhat common presentations.

Physical examination findings with Aspergillus sp. infection can include increased respiratory rate, increased pulmonary sounds, dyspnea, or other signs of pulmonary abnormalities. The patient may have only weight loss associated with chronic disease, as was the case in this patient. Common clinicopathologic abnormalities related to aspergillosis are variable. Birds frequently exhibit only a marked leukocytosis (usually with a heterophilia) with a total white blood cell count greater than 30,000 cells /µl. Monocytosis may or may not be present depending on the chronicity and location of the disease.

Lesions of aspergillosis often depend on the chronicity of the infection and the number of spores inhaled. Spores often grow on mucous membranes of the lungs and in air sacs, as with this parrot. Grossly, lesions include white-to-yellow plaques, which can be present in the trachea, syrinx, bronchi, or on air sacs and other serosal surfaces. Thickened air sacs with caseous exudates, caseous nodules in lung parenchyma with necrotic centers, and caseous nodules in other organ parenchyma can be observed. Cytologically, samples from lesions of aspergillosis contain necrotic debris with fungal hyphae characterized by thin parallel walls, septa, and branching. Inflammatory cells may include heterophils, macrophages, lymphocytes, and multinucleated giant cells. Infrequently, macroconidia containing fungal spores may be recovered from cavitiated lesions. Histologically, affected tissue may contain granulomatous foci and regions of caseous necrosis surrounded by epithelioid macrophages, multinucleated giant cells, heterophils, lymphocytes, and a connective tissue capsule. Special stains such as periodic acid-Schiff reaction or Gomori methanamine silver (GMS) staining may reveal the hyphae if they are not well visualized in hematoxylin and eosin-stained tissue sections.

Radiographs often display a loculated or cavitated appearance of air sacs, and/or increased opacity within the lung tissue. Direct visualization via endoscopy can further support the tentative diagnosis of aspergillosis. Ultimately, mycotic culture is required for definitive diagnosis.

Recently, there have been studies examining the efficacy of other diagnostic tests including psittacine-specific antigen and antibody ELISA tests, and plasma protein electrophoretograms (all available at University of Miami School of Medicine). Current ELISA tests do not appear to be highly sensitive screening tests for aspergillosis in psittacines. The electrophoretograms of birds with aspergillosis often result in increased concentrations of beta-globulins, a decreased concentration of albumin, and a decreased A/G ratio. However, while electrophoretograms result in more consistent findings with psittacines infected with aspergillosis, they can often have normal results in infected birds. Site, severity, and chronicity of infection can have some bearing on serologic and electrophoretic patterns. Serial antibody and antigen ELISA testing can be of use in some birds suspected of Aspergillus sp. infections. However, further research and studies are needed to improve the sensitivity and specificity of these diagnostic tests.

Experimental data in Cape shelducks suggest that a high and protective Aspergillus spp.-specific IgG titer can be attained with immunization. Further research still needs to be performed in the prophylaxis of fungal infection.

Therapy and Patient Outcome – Oral itraconazole (initially at 11 mg/kg BID, later decreased to SID) was instituted on 7/2/04 for 4-12 weeks, along with nebulization therapy with amphotericin B (50 mg reconstituted with 10 ml sterile water, further diluted in 6 ml sterile water and infused over 15-25 minutes) for 5 days BID starting on 7/2/04. The parrot initially weighed 269 grams. After several weeks of treatment, the bird weighed 304 grams. The bird remained on this therapy for approximately 2 weeks past radiographic resolution. The patient is currently eating well and acting normally.

References

1. Atasever A, Gumusoy KS: Pathological, clinical and mycological findings in experimental aspergillosis infections of starlings. J Vet Med 51:19, 2004.

2. Campbell TW: Avian Hematology and Cytology, 2nd ed., Iowa State University Press, Ames, 1995, pp. 59-62.

3. Graczyk TK, Cranfield MR, Klein PN: Value of antigen and antibody detection, and blood evaluation parameters in diagnosis of avian invasive aspergillosis. Mycopathologia 140:121-127, 1998.

4. Ivey ES: Serologic and plasma protein electrophoretic findings in 7 psittacine birds with aspergillosis. J Avian Med Surg 14:103-106, 2000.

5. Kearns KS: Avian Aspergillosis. Recent Advances in Avian Infectious Diseases. International Veterinary Information Service, Ithaca NY, 2003; A1902.0903.

6. Sakas PS: Essentials of Avian medicine: A Guide for Practitioners, 2nd ed., AAHA Press, Lakewood, 2002, pp. 90-92.

7. Schmidt RE, Reavill DR, Phalen DN: Pathology of Pet and Aviary Birds, Iowa State University Press, Ames, 2003, pp. 28-30, 36-38.

8. Tully TN, Lawton MP, Dorrestein GM: Avian Medicine. Butterworth-Heinemann, Woburn, 2000, pp. 45-49.

Acknowledgment

"Blue-Fronted Amazon Parrot" by Katherine Bell is from the Gallery of Bell Graphics website; it is copyrigted by Bell Graphics and is used with permission.

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