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Granulomatous myocarditis caused by candida albicans in a canary (serinus canaria).

Abstract: Candida albicans is among the major agents of mucous membrane mycosis in humans and animals, with systemic and deep infections observed in immunocompromised hosts. We describe a case of fatal granulomatous myocarditis caused by C albicans in a 20-day-old canary (Serinus canaria). The etiologic diagnosis was confirmed by identifying characteristic morphologic features of the organism, combined with histochemical staining, and followed by the use of ad hoc biomolecular analysis.

Key words: avian pathology, granulomatous myocarditis, histochemistry, molecular biology, polymerase chain reaction, Candida albicans, avian, canary, Serinus canaria

Clinical Report

A 20-day-old nestling canary (Serinus canaria) in poor body condition died spontaneously and was subsequently submitted for necropsy. The bird was part of a medium-sized flock of exhibition standard canaries that comprised 22 pairs of canaries of the Gloster Fancy breed, which were kept in a single room with an environmental temperature of 23[degrees]C-24[degrees]C (73.4[degrees]F 75.2[degrees]F) and a relative humidity of 75%-80%. Canaries were in the late breeding season and were fed with dried and sprouted seeds, lettuce (twice a week), and egg and biscuit mix and were supplemented with a multivitamin complex. Each pair of birds was placed in a breeding cage, the cages and perches were regularly cleaned, and the cage liner was replaced weekly. Because of a previous fowl pox outbreak, a specific vaccination program (Poulvac Canarypox FOI, Fort Dodge Animal Health SpA, Aprilia, Latina, Italy) had been planned and carried out. More recently, a cluster of air-sacculitis cases caused by Aspergillus species also was suspected in some birds. Consequently the entire flock was treated for 1 week with itraconazote (Itrafungol, Janssen Cilag SpA, Cologno Monzese, Milan, Italy).

On postmortem examination, the bird showed poor body condition, with reduced visceral fat stores and pectoral muscle atrophy. A single, whitish, round lesion 2 mm in diameter, which exhibited a smooth surface and a consistency softer than the surrounding tissue, was observed protruding from the epicardium at the level of the left ventricle (Fig 1). No other gross lesions were observed.

The entire heart was fixed in 10% neutral buffered formalin and embedded in paraffin. Tissues were sectioned in 5-[micro]n slices and were subsequently stained with hematoxylin and eosin, Gram's, Ziehl-Neelsen, periodic acid--Schiff, and Grocott methenamine-silver techniques. On microscopic examination, a granulomatous myocarditis was observed, with coalescing intralesional granulomas characterized by a necrotic core that showed no evidence of dystrophic calcification. These granulomas contained necrotic cell debris and were surrounded by epithelioid macrophages and large numbers of multinucleated giant cells (Fig 2). Grocott and periodic acid--Schiff stains showed the presence of several yeast, subspherical budding cells (up to 5 [micro]m in diameter), terminal and spherical chlamydospores (up to 10 [micro]m in diameter), and pseudohyphal elements. Yeast also were commonly observed within the cytoplasm of multinucleated giant cells (Figs 3 and 4). Results of Gram's and Ziehl-Neelsen stains were negative for bacteria. In the surrounding myocardium, the tissue was mildly atrophied, most likely secondary to compression.

[FIGURE 1 OMITTED]

To identify the causative agent, formalin-fixed and paraffin-embedded tissue sections were submitted for total DNA extraction and specific biomolecular analysis. After lysozyme digestion, DNA was purified with a QIAamp DNA mini kit (QIAGEN GmbH, Hilden, Germany). Fungal universal primers were used for polymerase chain reaction (PCR) amplification of the hypervariable D2 region (260 base pair [bp]) of the large subunit of the 25-28S rRNA gene. (1) Amplified PCR products were purified by using QIA quick PCR purification kit (QIAGEN) and directly sequenced by using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) in an Applied Biosystems (model 3100) automatic DNA sequencer. Finally, related DNA sequences were searched in GenBank by using the BLAST (Basic Local Alignment Search Tool) server (http://www.ncbi.nlm.nih.gov/ BLAST/). Our amplicons overlapped the corresponding sequences of Candida albicans strain DV17 (accession number GQ495089.1, 100% for 260 bp) and strains CBS 562 and CBS 1905 (accession number AY497682.1, 100% for 260 bp; and AY497673.1, 100% for 259 bp). On the basis of the above findings, a diagnosis of granulomatous myocarditis caused by C albicans was made.

Discussion

This report confirms C albicans as the cause of a fatal granulomatous myocarditis in a canary. Members of the genus Candida, particularly C albicans, are commensal dimorphic fungi that inhabit the skin as well as the upper-respiratory, alimentary, and genital tracts of humans and ectotherm animals, including birds. (2) Candida albicans is one of the major mycotic agents that affect both mucous membranes and the urogenital tissues. This organism also is responsible for deep infections in individuals with an impairment of either natural or acquired immunity. (3) Candida albicans also may be regarded in birds as an opportunistic yeast that causes a variety of disease conditions that involve the digestive tract and is frequently involved in ingluvitis. Furthermore, C albicans also has been documented as the causative organism of primary disease in many avian species. (2) In-breeding and line-breeding techniques for unusual color patterns may create less genetically resistant birds, which may be a contributing factor to a decline in resistance to disease in the smaller pet bird species, as in this case. (4) Additional predisposing factors may include immunosuppressive conditions, such as steroid administration, prolonged antibiotic therapy, subclinical malnutrition (particularly vitamin A deficiency), and viral infections. Psittacine birds seem to be highly susceptible to Candida infection, although pigeons and poultry raised under suboptimal conditions also are at risk. (2,4) Although C albicans is most often responsible for disease in pet birds, Candida parapsilosis and Candida tropicalis also have been cultured from lesions in affected birds. (4,5) Systemic or deep Candida infections are rare and usually develop in severely debilitated birds, with Candida being isolated from the blood, bone marrow, or parenchymatous organs of animals with generalized disease. Systemic candidiasis in a flock of canaries was previously found associated with central nervous system signs, as well as typical gastrointestinal lesions, characterized by pseudomembranous necrotic patches that were easily peeled from the eroded mucosal surface. (4)

Cardiac fungal infection (CFI) is relatively uncommon in humans and animals, although its incidence has been reported to be increasing in human patients. Malignant neoplasms, AIDS, hematologic disorders, and the prolonged use of antibiotics or corticosteroids are frequently reported as underlying conditions. (6) Candida is the leading pathogen that causes CFI in humans; nevertheless, increasing frequencies of infection due to Aspergillus and Mucor species also have been reported. The reason why noncandidal CFIs are also increasing in frequency is unclear, although this may reflect recent changes in clinical settings, such as the hospital environment, the availability of more powerful and broad-spectrum antimicrobial agents, and an increase in the number of patients with underlying conditions characterized by greater morbidity. (6)

[FIGURE 2 OMITTED]

Mycotic infections of the heart also are infrequent in pet birds; these infections usually are the result of disease extension from air sacs. However, disseminated fungal infections can develop in immunocompromised hosts, which result in hematogenous dissemination to the heart. (7) Restrictive pericarditis with secondary cardiomyopathy attributed to Candida species was reported in a sun conure (Aratinga solstitialis). (8) In the canary we describe the apparent lack of air sac and serosal involvement would argue in favor of a hematogenous infection, presumably involving the coronary vessels that supply the epicardium. Nevertheless, the possibility that preexisting lesions may have been "mitigated" (or may have even "disappeared") after antifungal drug administration cannot be ruled out. Age likely played a crucial predisposing role in this bird; indeed, young animals are known to be particularly susceptible to spontaneous candidiasis, possibly because of an immature immune system or an incompletely developed gastrointestinal defense system. (2) However, the concurrent role of additional intrinsic factors (resulting from genetic selection) should also be considered.

[FIGURE 3 OMITTED]

Because the antemortem diagnosis of CFI is very difficult, studies performed on necropsy specimens are extremely important to gain a better knowledge of this pathologic condition. To this aim, the presence of fungal hyphae should be demonstrated, but a specific etiologic diagnosis would require that the organism be cultured. (7) Although yeast species are routinely defined according to cellular morphology and distinctive reactivity in a standard set of fermentation and assimilation tests, molecular comparisons have shown that phenotypic features are sometimes unreliable for this specific aim. Even today, methods based on phenotypic analysis occasionally generate misidentification. Furthermore, the frequent recovery of uncommon yeast from clinical specimens, with special reference to immunocompromised patients, highlights the need for broad-range identification methods. In this respect, biomolecular tests that rely on direct screening of PCR products represent a powerful tool for fungal identification, (1) an option that also is feasible on formalin-fixed, paraffin-embedded tissues. The biomolecular techniques used in this case are useful diagnostic tools that allow detailed identification of this fungal agent from formalin-fixed, paraffin-embedded tissue samples.

[FIGURE 4 OMITTED]

References

(1.) Putignani L, Paglia MG, Bordi E, et al. Identification of clinically relevant yeast species by DNA sequence analysis of the D2 variable region of the 25-28S rRNA gene. Mycoses. 2008;51 (3):209-227.

(2.) Bauck L. Mycoses. In: Ritchie BW, Harrison GJ, Harrison LR, eds. Avian Medicine. Principles and Application. Lake Worth, FL: Wingers Publishing; 1994:998-1006.

(3.) Brown CC, Baker DC, Barker IK. Mycotic diseases of the gastrointestinal tract. In: Maxie MG, ed. Jubb, Kennedy, and Palmer's Pathology of Domestic Animals. Vol. 2. 5th ed. Philadelphia, PA: Saunders Elsevier; 2007:229-231.

(4.) Velasco MC. Candidiasis and cryptococcosis in birds. Semin A vian Exotic Pet Med. 2000;9(2):75-81.

(5.) Joseph V. Infectious and parasitic diseases of captive passerines. Semin Avian Exotic Pet Med. 2003;12(1): 21-28.

(6.) Chinen K, Tokuda Y, Sakamoto A, Fujioka Y. Fungal infections of the heart: a clinicopathologic study of 50 autopsy cases. Pathol Res Pract. 2007; 203(10):705-715.

(7.) Smith RE, Reavill DR, Phalen DN. Cardiovascular system. In: Smith RE, Reavill DR, Phalen DN, eds. Pathology of Pet and Aviary Birds. Ames, IA: Blackwell Publishing; 2003:1-16.

(8.) Smith RE. Avian diseases that antibiotics won't cure: from Eastern States Veterinary Conference. AAV Today. 1987; 1(2):69-70,72.

Giuseppe Marruchella, DVM, Gianluca Todisco, DVM, Silvia D'Arezzo, BS, Giovanni Di Guardo, DVM, Dipl ECVP, and Maria Grazia Paglia, BS

From the Department of Comparative Biomedical Sciences, Faculty of Veterinary Medicine, University of Teramo, Piazza Aldo, Moro 45, 64100, Teramo, Italy (Marruchella, Todisco, Di Guardo); and the National Institute for Infectious Diseases "Lazzaro Spallanzani," Molecular Microbiology, Via Portuense 292, 00149, Rome, Italy (D'Arezzo, Paglia).
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Article Details
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Title Annotation:Clinical Reports
Author:Marruchella, Giuseppe; Todisco, Gianluca; D'Arezzo, Silvia; Di Guardo, Giovanni; Paglia, Maria Grazi
Publication:Journal of Avian Medicine and Surgery
Article Type:Report
Geographic Code:4EUIT
Date:Sep 1, 2011
Words:1754
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