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History

A 7-year-old female blue-headed pionus parrot (Pionus menstruus) was presented to the University of Illinois Veterinary Teaching Hospital (Urbana, IL, USA) for poor body condition. The bird was part of a breeding pair housed in an indoor/ outdoor facility and in the same room as several other breeding pairs of parrots, including Quaker parakeets (Myiopsitta monachus), Amazon parrots (Amazona species), and sun conures (Aratinga solstitialis solstitialis). The bird had been obtained from another breeder approximately l year before presentation. The owners had been away for 3 months during which time a caretaker was managing the pionus parrot; therefore, the duration of weight loss was not known. No illness or problems were observed before the owner's absence. Three days before the owners arrived home, one of the Quaker parakeets was found dead in the cage. A necropsy was not performed and no additional information was available regarding the dead bird. The typical diet for the pionus parrot consisted of seed mix, fresh fruits, greens, beans, and other vegetables; though the owners were unsure if the bird was receiving anything other than the seed mix while they were gone. On examination the pionus parrot was quiet, alert, responsive, and mildly dyspneic. Crusting was observed around the nares, and the choanal papillae were blunted. The animal weighed 214 g and was severely cachectic. A large, firm, approximately 3-4-cm mass incorporating most of the caudal coelomic cavity was palpable. The bird's droppings and feathers appeared normal.

The initial diagnostic plan for this case was to collect baseline hematologic and radiographic data. A blood sample was collected from the right jugular vein for a complete blood count (CBC) and plasma biochemical profile. Results showed the bird was anemic (hematocrit, 29%; reference range, 35%-45%). (1) Although results of the total white blood cell count were within the reference range, a left shift with band cells (0.4 x [10.sup.3] cells/[micro]l) (1) and a monocytosis (0.6 x [10.sup.3] cells/[micro]l reference range, 0-230 cells/[micro]l) were present. (1) Abnormal results of the plasma biochemical analysis were a high concentration of aspartate aminotransferase (AST) (692 U/L; reference range, 135-365 U/L) (1) and creatine kinase (CK) (1074 U/L; reference range, 100-300 U/L), (2) hyperglycemia (590 mg/dl; reference range, 125 300 mg/dl), (1) hypophosphotemia (2.5 mg/dl; reference range, 2.9-6.6 mg/dl), (1) hyperkalemia (5.7 meq/L; reference range, 3.5-4.6 meq/L), (1) and hyponatremia (141 meq/L; reference range, 145-155 meq/L). (1) Whole body ventrodorsal (Fig 1) and lateral (Fig 2) radiographs were taken with light restraint.

After reviewing results of the hematologic tests and radiographs, a fine-needle aspirate of the coelomic mass was collected. The cytologic sample was prepared with Wright-Giemsa stain and evaluated by light microscopy (Fig 3).

[FIGURE 1 OMITTED]

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[FIGURE 3 OMITTED]

At this time, please review the history, examination findings, results of hematologic and plasma biochemical profiles, radiographs (Figs 1 and 2), and cytologic smear (Fig 3) and form a list of differential diagnoses before proceeding.

[FIGURE 4 OMITTED]

Diagnosis

Results of the CBC showed a mild nonregenerative anemia, which was likely caused by chronic disease. The left shift with a monocytosis indicated chronic active inflammation. The high concentrations of AST and CK were likely caused by muscle degeneration evident in the patient's body condition. The AST concentration may have also been high because of hepatic cellular damage. The high potassium level may have been associated with cellular degeneration. Radiographic findings showed the ventriculus was displaced caudally and to the left by a large mass effect in the cranial cavity and right portion of the coelomic cavity. Differential diagnoses for these findings include hepatomegaly with the possibility of free fluid, abscess, granuloma, and neoplasia.

The cytologic sample was prepared with Wright-Giemsa stain and evaluated under light microscopy (Fig 3). The cytologic sample was highly cellular and consisted of numerous macrophages filled with negative-staining, rod-shaped bacteria, estimated at less than 1 [micro]m in length. The bacteria were also identified as extracellular in the cytologic sample. There were rare clusters of hepatocytes, some of which contained clear, round, and variably sized vacuoles suggestive of lipid. Numerous red blood cells and several white blood cells were observed in the background, consistent with peripheral blood contamination. The cytologic interpretation was marked granulo matous inflammation with intracellular negative-staining bacteria consistent with Mycobacterium species. The organisms were positive on Ziehl-Neelsen staining. The rare hepatocytes observed in the sample smears supported a liver aspirate.

With the cytologic findings being consistent with mycobacteriosis and the poor prognosis due to mass effect, emaciation, and dyspnea, the owner elected euthanasia. The carcass was submitted for necropsy and speciation of the bacterium to determine risk to the rest of the flock and their caretakers.

On gross necropsy, hepatomegaly, splenomegaly, and a thickened and cloudy appearance to the air sacs (Fig 4) were observed. On histopathologic examination, a granulomatous inflammatory infiltrate was found in multiple tissues. Approximately 70% of the liver, 80% of the spleen, and 50% of the bone marrow parenchyma was replaced by a massive infiltration of large macrophages and multinucleated giant cells that contained many acid-fast bacilli. The small and large intestines segmentally contained multifocal to coalescing infiltration of similar cells within the lamina propria and submucosa, resulting in villous blunting and fusion as well as gland and crypt loss. The ventricular serosal surface and adjacent mesentery, the cervical stroma surrounding the trachea and esophagus, and sections of air sac membranes all contained a similar infiltrate; the cervical stroma and air sacs also contained variable numbers of heterophils, lymphocytes, plasma cells, fibrin, and proteinaceous edema. The lungs contained rare small to moderate-sized foci of macrophages and multinucleated giant cells. No histopathologic abnormalities were found in brain, heart, kidneys, trachea, esophagus, crop, or pancreas. Samples for aerobic culture were plated on Columbia blood agar and MacConkey agar (Remel, Lenexa, KS, USA) and incubated a minimum of 48 hours at 37[degrees]C with 5% carbon dioxide. Samples for anaerobic culture were plated on Brucella agar, kanamycin-vancomycin-laked blood agar, and phenylethyl alcohol agar with sheep blood (Remel) and incubated a minimum of 5 days at 37[degrees]C in an anaerobic chamber. No growth was found on either aerobic or anaerobic bacterial culture. Acid-fast staining (Ziehl-Neelsen) of multiple tissues revealed rod bacteria consistent with a Mycobacterium species. A definitive diagnosis was made by polymerase chain reaction (PCR) rRNA sequencing (primers listed in Table 1) using published protocols. (3) The sequences were compared with GenBank (National Center for Biotechnology Information, GenBank; www.ncbi.nlm.nih.gov) using BLAST software. (4) Results were 100% consistent with Mycobacterium avium subsp avium.

Comments

Mycobacterium species are aerobic, non-sporeforming, nonmotile acid-fast rods that are resistant to desiccation and therefore difficult to clear from the environment. The most commonly diagnosed organisms associated with psittacine mycobacteriosis are M avium or Mycobacterium genavense. (5) Though rare, cases of Mycobacterium tuberculosis have been documented in birds. (6) Mycobacterium avium subsp avium is considered ubiquitous. (5,7) Mycobacterium avium is shed in the feces and urine of infected birds and spread through the environment in soil or water or by aerosolized feces. Although a zoonotic potential exists, generally involving only the young, old, or immunocompromised individuals, to our knowledge there are no reports of transmission of M avium subsp avium from birds to humans. (5,6) Affected birds may present for cessation of egg laying, chronic wasting despite a good appetite, recurrent diarrhea, polyuria, anemia, and dull plumage. There are 3 commonly recognized forms of avian mycobacteriosis: 1) the classic form, with tubercles or granulomas diffusely throughout the body; 2) the paratuberculosis form, with lesions present in the intestinal tract; and 3) the nontuberculous form, which is hard to recognize at necropsy because of the lack of gross lesions and granulomas. (5) The paratuberculosis form is the most common form seen in Amazon, pionus, brotogeris, and psittacula parrots? Infections are usually initiated via ingestion of the bacteria, leading to infection affecting the intestinal tract. (6) Once the organism has invaded the intestinal tract, a subclinical bacteremia occurs with hematogenous spread to the liver and other organs. Granuloma formation can occur in any organ but is generally localized to the intestinal tract and reticuloendothelial organs. (5,8,9) Granulomas may remain dormant for a prolonged time and then become active when the host becomes immunosuppressed. Birds are also susceptible to Mycobacterium species infections by the respiratory tract. Inhalation generally causes lesions in the lungs and air sacs. (5,8,9) Although less common, it is possible to see cutaneous lesions from wounds or punctures. (10)

Antemortem diagnosis of mycobacteriosis can be very challenging. A definitive diagnosis is often made by pursuing a parallel testing strategy, including a CBC (severe leukocytosis), radiographic evaluation with evidence of granulomas or hepatomegaly, fecal cytology demonstrating the presence of acid-fast bacteria, and culture and/or PCR testing of the feces, sputum, or tissues. Acid-fast staining may be used to characterize Mycobacterium species to the generic level but cannot be used differentiate the specific species or subspecies causing the infection. (11) Endoscopic examination and biopsy of abnormal tissues or granulomas with confirmation by PCR testing, histopathologic examination, or culture is preferred. Mycobacterial culture, historically considered the gold standard for diagnosis, generally requires prolonged incubation periods because of the fastidious nature of some Mycobacterium species. (11) Testing by PCR has become a common method to identify the more fastidious M genavense (11, 12) and is replacing culture as the gold standard diagnostic test for all Mycobacterium species represented in the GenBank database (National Center for Biotechnology Information). This method can be performed on any tissue demonstrating acid-fast bacteria and has been used on a wide variety of species including mammals, birds, (12) amphibians, and fish. Most large veterinary diagnostic laboratories offer 16S rRNA gene sequencing as a diagnostic test for mycobacteriosis at a reasonable cost ($100). Furthermore, conclusive results may be obtained rapidly compared with culture, which may require many weeks to months to complete. Positive and negative PCR controls as well as a control for quality of DNA extraction are run with each sample. Validation of the characteristic size amplicons produced by using Mycobacterium-specific primers is by sequence analysis showing significant homology when compared with Gen Bank or other published Mycobacterium sequences. Serologic techniques provide a less invasive method for diagnosis but are still being developed. (7) Definitive diagnosis is often made based on lesions seen at necropsy and histopathologic results demonstrating the organism, as well as on culture and PCR results.

Although treatment for avian mycobacteriosis is available, euthanasia is generally recommended because of the potential risk to other animals, the severity of disease usually present at the time of diagnosis, and the zoonotic potential. If treatment is pursued, long-term quarantine (minimum of 2 years) of flock birds and routine fecal examination every 6-12 weeks should be implemented. Medications with antimycobacterial activity include azithromycin, isoniazid, enrofloxacin, clarithromycin, doxycycline, ciprofloxacin, rifabutin, rifampicin, amikacin, clofazimine, and ethambutol. Longterm multi-drug therapy should be used because of the bacterium's slow growth and potential for drug resistance. The environment should be cleaned regularly with 5% phenol or 5% chlorine bleach regularly to help decontamination. There have been a few reports of successful treatment of avian mycobacteriosis5 but it is unknown what overall percentage of cases these represent.

The blue-headed pionus is one of the psittacine species considered to be more prone to develop mycobacteriosis. (5) Most cases follow a typical presentation characterized by chronic disease, diarrhea, and severe leukocytosis (5); however, the bird in this case had an atypical presentation. The initial differentials for diagnosis include dystocia or neoplasia, with early testing supportive of a tumor. The absence of a typical profound leukocytosis may be because of effacement of the bone marrow after colonization by the organism following hematogenous spread. Because of the severe infiltration of the intestines by the bacteria and granuloma formation causing malabsorption, birds with mycobacteriosis secondary to ingestion of the bacteria typically develop diarrhea. This bird only had segmental infiltration of the intestines, which was not extensive enough to cause a clinical change in the feces.

Mycobacteriosis should be considered in any avian patient presenting in poor body condition, normal appetite, and having a coelomic mass effect, especially if there is a history of environmental exposure. The diagnosis of M avium subsp, avium has significant consequences for owners and breeders including possible zoonosis and ethical considerations in the sale of animals that have been exposed.

This case was submitted by Anne BurgdorfMoisuk, DVM, Julia K. Whittington, DVM, Shir Gilor, MSc, DVM, and David Coleman, MS, DVM, Department of Veterinary Clinical Medicine, University of Illinois College of Veterinary Medicine, (Burgdorf-Moisuk and Whittington), 1008 W Hazelwood Dr, Urbana, IL 61802, USA; and the Veterinary Diagnostic Laboratory, University of Illinois College of Veterinary Medicine (Gilor and Coleman), 2001 S Lincoln Ave, Urbana, IL 61802, USA.

References

(1.) Pollock CP, Carpenter JW, Antinoff N. Birds. In: Carpenter JW, ed. Exotic" Animal Formulary. 3rd ed. St Louis, MO: Elsevier Saunders; 2005:268.

(2.) Harrison G J, Lightfoot TL. Clinical Avian Medicine. Vol II. Palm Beach, FL: Spix Publishing Inc; 2006.

(3.) Park H, Jang H, Kim C, et al. Detection and identification of mycobacteria by amplification of the internal transcribed spacer regions with genus-and species-specific PCR primers. J Clin Microbiol. 2000;38:4080-4085.

(4.) Altschul SF, Madden TL, Schaffer AA, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389-3402.

(5.) Lennox AM. Mycobacteriosis in companion psittacine birds: a review. J Avian Med Surg. 2007;21: 181-187.

(6.) Hoop RK. Mycobacterium tuberculosis infection in a canary (Serinus canaria L.) and a blue-fronted Amazon parrot (Amazona amazona aestiva). Avian Dis. 2002;46:502-504.

(7.) Gray PL, Saggese MD, Phalen DN, Tizard I. Humoral response to Mycobacterium avium subsp. avium in naturally infected ring-neck doves (Streptopelia risoria). Vet Immunol Immunopathol. 2008; 125:216-224.

(8.) Heatley JJ, Mitchell MM, Roy A, et al. Disseminated mycobacteriosis in a bald eagle (Haliaeetus leucocephalus). J Avian Med Surg. 2007;21:201-209.

(9.) Gerlach H. Bacteria. In: Ritchie BW, Harrison G J, Harrison LR, eds. Avian Medicine." Principles and Application. Lake Worth, FL: Wingers; 1994:949-983.

(10.) Tell LA, Woods L, Cromie RL. Mycobacteriosis in birds. Rev Sci Tech. 2001;20:180-203.

(11.) Tell LA, Foley J, Needham ML, Walker RL. Comparison of four rapid DNA extraction techniques for conventional polymerase chain reaction testing of three Mycobacterium spp. that affect birds. Avian Dis'. 2003;47:1486-1490.

(12.) Tell LA, Leutenegger CM, Larsen RS, et al. Real-time polymerase chain reaction testing for the detection of Mycobacterium genavense and Mycobacterium avium complex species in avian samples. Avian Dis. 2003;47:1406-1415.
Table 1. Primers used for polymerase chain reaction
rRNA sequencing of hepatic tissues from 7-year-old
female blue-headed pionus parrot that presented with a
coelomic mass and cachexia.

     Primer                   Sequence

Generic fungal
  18S ITS4                  5'-TCC TCC GCT TAT TGA TAT GC-3'
  18S ITS5                  5'-GGA AGT AAA AGT CGT AAC AAG G-3'
Mycobacterium species
  Mycobacterium ITSF        5'-TGG ATC CGA CGA AGT CGT AAC AAG G-3'
  Mycobacterium mycom 2     5'-ATG CTC GCA ACC ACT ATC CA-3'
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Publication:Journal of Avian Medicine and Surgery
Article Type:Report
Geographic Code:1USA
Date:Sep 1, 2009
Words:2536
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