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A Muscovy duckling was presented for necropsy due to ongoing mortalities on a farm. Microscopic examination revealed multisystemic inflammatory lesions with intralesional and intracytoplasmic yeast-like organisms. We identified these agents as yeast belonging to the Order Saccharomycetales based on sequence data obtained from the ribosomal RNA operon.
Infection disséminée à levures (Ordre Saccharomycetales) chez un caneton musqué (Cairina moschata). Un caneton musqué a été présenté pour la nécropsie en raison de mortalités se produisant à une ferme. Un examen microscopique a révélé des lésions inflammatoires multisystémiques par des organismes intralésionnels et intracytoplasmiques s’apparentant à des levures. Nous avons identifié ces agents comme des levures appartenant à l’ordre Saccharomycetales en nous fondant sur des données obtenues auprès de l’opéron ARN risbosomique.
(Traduit par Isabelle Vallières)
Mortalities associated with numerous intracellular organisms in the capillary endothelial cells of different body systems in Muscovy (Cairina moschata) and domestic (Anas platyrhynchos domestica) ducks have been reported from Canada and the United Kingdom, but the identity of these organisms and their classification remains undetermined (1,2). Previous studies described these intracellular organisms in Muscovy duck disease as protozoa (3), probably bacteria capable of forming spores (2), and, most recently, unusual intracellular periodic acid–Schiff (PAS) positive budding organisms (1). Recently, morphologically similar organisms were observed in the tissues of a blue heron and identified as yeast belonging to the Order Saccharomycetales (4). Here, we report a case of disseminated infection and associated pathological lesions in a Muscovy duckling with similar intracellular organisms identified as yeast belonging to the Order Saccharomycetales.
A 2-month-old farm-raised Muscovy duckling was submitted for necropsy by a referring veterinarian to the pathology laboratory of Alberta Agriculture and Forestry in Edmonton. The duckling was part of a group of 11 birds hatched in early August 2013, some of which may have been hybrid Muscovy/Indian Runner ducks. Approximately 1 wk after hatching, the ducklings were relocated to a large open slough on the farm. Six ducklings died during September and October before they were fully fledged. Clinical signs included acute illness with lethargy and rapid progression to death. The referring veterinarian performed a postmortem examination on 1 dead bird, found no significant gross lesions and conducted no further tests. Due to ongoing mortality, a fresh dead duckling was submitted for diagnostic investigation, and was found to be in good body condition with diffusely congested and edematous lungs and spleen that was enlarged nearly 2 times the normal size.
Portions of brain, heart, trachea, lungs, liver, spleen, kidneys, small and large intestines, and sciatic nerves were fixed in 10% neutral buffered formalin. Tissue specimens were processed routinely for histology, and 5-μm thick sections were stained with hematoxylin and eosin (H&E). Histologically, there was a severe, non-suppurative interstitial pneumonia with clusters of round to oval, slightly basophilic intracellular organisms measuring approximately 2 μm × 0.5 to 1.0 μm in the cytoplasm of blood capillary endothelial cells and macrophages in the atrial septa which connect the atrial lumens with the parabronchial lumens (Figure 1A). Air capillary interstitium was expanded and infiltrated with small numbers of lymphocytes, plasma cells, and macrophages. Para-bronchi and atria were filled with eosinophilic proteinaceous edema. Intracellular organisms in endothelial cells and macrophages with similar inflammatory infiltrate were also present in the tracheal mucosa, interstitium of myocardium, exocrine pancreas, spleen, mucosa and submucosa of the small intestine, choroid plexus (Figure 1B) and sciatic nerve (Figure 1C).
To further characterize the morphology of intracellular organisms, Periodic acid-Schiff (PAS), Ziehl–Neelsen (ZN) acid-fast, modified Brown and Brenn (BB) Gram stain and Grocott’s methenamine silver (GMS) stained sections were prepared from paraffin-embedded formalin-fixed lung tissues. The intracytoplasmic organisms had weak Gram-negative staining. The cell walls of the intracellular organisms stained positively with PAS stain indicating the presence of glycogen as seen in fungi, and did not stain with ZN. The GMS stain revealed argyrophilic, intracytoplasmic, budding, yeast-like organisms (Figure 1D).
Routine bacterial culture on liver and spleen, and polymerase chain reaction (PCR) testing for avian influenza (AI) matrix and avianparamyxovirus-1 (APMV-1) were performed on tracheal tissue. No attempt at fungal culture was made at the time. The PCR testing for AI and APMV-1 was negative and bacterial culture results were considered to be not significant.
DNA was extracted from formalin-fixed paraffin-embedded lung tissue using a commercial kit (QIAamp DNA FFPE Tissue Kit; Qiagen, Mississauga, Ontario). The PCR reactions were performed using primers designed to amplify a segment of the rRNA operon of the uncultured yeast previously detected in lung tissue of a great blue heron (Ardea herodias) found dead in Moose Jaw, Saskatchewan, Canada (GenBank accession FJ848337) (4). Primers JH0103 (5′-GGA AAC TCA CCA GGT CCA GA-3′) and JH0105 (5′-AAG CAT CGC GAT TCC ATA AA-3′) amplify an 817 bp region of the genome containing partial 18S rRNA gene, internal transcribed spacer 1, the 5.8S rRNA gene and partial internal transcribed spacer 2. The PCR product of the expected size was obtained from the lung DNA extract. The PCR amplicons were sequenced directly using the amplification primers, and an internal primer, JH0104 (5′-CTT GCG TTG ATT ACG TCC CT-3′). The sequence of this amplicon was 96% identical over 804 bp to the rRNA operon sequence identified in the previous great blue heron case (GenBank accession FJ848337). Phylogenetic analysis supported the placement of the organism detected in the duckling lung tissues as a member of the Saccharomycetales Order of yeasts (5). No other sequences were identified with > 83% identity to the query sequence. The partial rRNA operon sequence from the duckling was deposited in GenBank (Accession KM103295).
Histopathological lesions with multisystemic intralesional and intracellular yeast organisms, and results of PCR investigation in this case support disseminated yeast infection as the likely cause of acute illness and death of this duckling. The intracellular yeast organisms were similar in tissue distribution and morphology to the intraendothelial organisms described previously in farmed Muscovy and domestic ducks in Canada and the United Kingdom (1–3). In all these cases, mortality occurred between August and December and was typically associated with history of access to a pond. The reported clinical signs in the previous cases range from ailing for a day or so to lameness, weakness, ataxia, respiratory distress, and rapid death. Histological features in the aforementioned cases included interstitial pneumonia, myocarditis, hepatitis, and splenitis with many prominent 1 to 2 μm intraendothelial organisms predominantly in lung tissues similar to the present case, and fewer organisms in other organs. Morphologically similar intraendothelial organisms with the absence of significant inflammatory lesions in multiple organs (predominantly lungs) were reported from a great blue heron (Ardea herodias) that was found dead in Moose Jaw, Saskatchewan, suggesting that wild birds may act as carriers of the yeasts (4). The organisms that were found in the great blue heron were identified as yeast of the Order Saccharomycetales based on ultrastructural morphology and sequence data from the ribosomal RNA operon. The PCR testing and gene sequencing of the RNA obtained from organisms seen in the lung tissues in this case revealed 96% identity to the yeast identified in the previous great blue heron.
Various bacterial and viral infections are reported to cause mortalities in ducklings. Duck viral enteritis (DVE) is a highly contagious disease characterized by acute death with high mortality in Muscovy ducks and infected ducks have hemorrhages and necrosis of internal organs with intranuclear viral inclusions in multiple body systems (6,7). Muscovy duck parvovirus (MDPV), another viral infection, is reported to cause weakness with pale thigh/leg muscles and myocardium, fibrinous exudate on the liver capsule, ascites, and mortality in Muscovy ducks (8–10). Ducks infected with duck circovirus (DuCV) have histological evidence of necrosis of lymphoreticular tissues and viral inclusions which can lead to immunosuppression and increased susceptibility to secondary infections (11). Duck reovirus (DRV) infection in Muscovy ducks causes necrotizing lesions in the spleen, bursa of Fabricius, liver, and myocardium, and subsequent immunosuppression resulting in secondary infections and eventual mortality (12,13). Duck hepatitis virus (DHV) type I is a fatal virus infection of young ducklings characterized by multisystemic hemorrhages, hepatitis, pancreatitis, and encephalitis (14). The gross and histological lesions seen in this case were not consistent with any of the above viral infections or bacterial septicemia.
The disease in Muscovy ducks caused by yeast appears to be strongly associated with aquatic environments, and occurs following access to ponds that were also visited by other wild birds (1–3). Reports indicated that wild and migratory birds carry various yeasts and contribute to environmental contamination through fecal shedding (15–17). Many aquatic bird species including American white pelicans (Pelecanus erythrorhynchos), lesser scaup (Aythya affinis), and Pekin ducks (Anas platyrhynchos domestica) in Saskatchewan have morphologically similar but unidentified organisms without inflammation in their tissues (G. Wobeser, unpublished data, 1990). In our case, since mallards (Anas platyrhynchos), teals (Anas crecca), Canada geese (Branta canadensis), and muskrats (Ondatra zibethicus) co-mingled with Muscovy ducks on the same pond, the pond environment had a high likelihood of being contaminated by the yeast shed by wild birds.
This report describes disseminated infection and mortality in a Muscovy duckling associated with novel invasive yeast belonging to the Order Saccharomycetales. Yeasts of the Order Saccharomycetales belong to Phylum Ascomycota and comprise about 1000 known Ascomycetes species. These yeasts live as saprobes, often in association with plants, animals, and their interfaces (18). A few of these species account for opportunistic fungal infections in humans and animals (19–23). Wild birds which harbor some of these yeast species as normal inhabitants in their gastrointestinal tract can pose a risk to the health of humans and animals through fecal shedding and environmental contamination (15–17,24–28). Pathogenesis of Muscovy Duck Disease caused by these yeast organisms is unknown. We hypothesize that the duckling in this report was exposed orally or through the respiratory route to the yeast stages in the pond environment which led to disseminated infection and mortality. The underlying factors that led to increased susceptibility of yeast infection in this case are unknown, but poor immunity from immaturity or other undetermined immune suppressors is a possibility. The prevalence, pathogenesis, and impact of this type of yeast infection in wild duck populations are unknown. It appears that this novel yeast species can cause sporadic and local mortality events in Muscovy ducklings, and fungal infections should be considered as a differential diagnosis for acute illness in ducklings with nonspecific gross postmortem findings.
The authors gratefully acknowledge the staff of the Animal Health and Assurance Division of Alberta Agriculture and Forestry (AAF) for supporting this investigation. The authors thank Dr. Eryn Hanak for case submission, Dr. Jagdish Patel for suggestions during preparation of this manuscript, and Lisa Zeldenrust for preparing histology slides. CVJ
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