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Journal of Veterinary Diagnostic

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 DOI: 10.1177/104063871002200222

2010 22: 282J VET Diagn InvestBarr

Arno Wünschmann, Daniel Rejmanek, Patricia A. Conrad, Natalie Hall, Luis Cruz-Martinez, Samuel B. Vaughn and Bradd C. Infections in Free-Ranging Eagles in North AmericaSarcocystis FalcatulaNatural Fatal

  

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CASE REPORTS

Natural fatal Sarcocystis falcatula infections in free-ranging eagles in North America

Arno Wunschmann,1 Daniel Rejmanek, Patricia A. Conrad, Natalie Hall, Luis Cruz-Martinez,Samuel B. Vaughn, Bradd C. Barr

Abstract. Three bald eagles (Haliaeetus leucocephalus) and 1 golden eagle (Aquila chrysaetos) wereadmitted to rehabilitation facilities with emaciation, lethargy, and an inability to fly. Intravascular schizontsand merozoites were present in 2 bald eagles, mainly in the lung tissue, whereas the third bald eagle and thegolden eagle had lymphohistiocytic encephalitis with intralesional schizonts and merozoites. In all eagles,protozoal tissue cysts were present in skeletal musculature or heart. The protozoal organisms weremorphologically compatible with a Sarcocystis sp. By immunohistochemistry, the protozoal merozoites werepositive for Sarcocystis falcatula antigen in all cases when using polyclonal antisera. Furthermore, theprotozoa were confirmed to be most similar to S. falcatula by polymerase chain reaction in 3 of the 4 cases. Tothe authors’ knowledge, this report presents the first cases of natural infection in eagles with S. falcatula as acause of mortality.

Key words: Aquila chrysaetos; bald eagles; electron microscopy; golden eagle; Haliaeetus leucocephalus;immunohistochemistry; polymerase chain reaction; Sarcocystis falcatula.

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Sarcocystis falcatula is a protozoan that inhabits theintestine of the Virginia opossum (Didelphis virginiana),which is its definitive host in North America. Thus far,members of 4 orders of birds, namely Psittaciformes,Passeriformes, Columbiformes, and most recently Strigi-formes have been recognized as intermediate hosts for thisparasite.3,4,6,11,22,24,26 Neuronal infection by Sarcocystisorganisms usually is attributed to Sarcocystis neurona,another intestinal sarcocyst of the Virginia opossum.However, S. falcatula also is capable of infecting neuronsin birds.26 This study documents, for the first time, thatgolden eagles and bald eagles (order Falconiformes) mayserve as intermediate hosts for S. falcatula and that S.falcatula can cause clinical disease and death in thesespecies from encephalitis with neuronal infection or fromwidespread pulmonary endothelial cell infection.

Case 1 involved an adult female golden eagle (Aquilachrysaetos). Cases 2, 3, and 4 involved an adult female baldeagle (Haliaeetus leucocephalus), an adult male bald eagle,and a second-year male bald eagle, respectively. The birdswere unable to fly and were admitted to rehabilitationfacilities in Virginia, Indiana, and Minnesota. The goldeneagle and 2 bald eagles (cases 1, 2, and 3) were hospitalized

for 3–4 days before they died despite supportive care. Onebald eagle (case 4) was euthanized at admission because ofan inoperable wound with metallic projectile fragmentsexposing the joint surfaces of the tarso-metatarsal jointdespite being bright, alert, and responsive. All hospitalizedbirds were emaciated, dehydrated, anemic, and hypopro-teinemic (Table 1). The golden eagle exhibited mioticpupils, and consensual reflexes were absent. Placing deficitswere present in both limbs, and grasp reflexes weremarkedly weakened bilaterally. Toe pinch reflex and wingreflexes were diminished. The eagle had intention tremorswhen attempting to apprehend food. The bird exhibitedlabored breathing and died. One bald eagle (case 2) washock sitting on initial presentation, with a mild bilateralwing droop and mild ventroflexion of the neck. Within48 hr of admission, the patient began exhibiting a normalstance but became dyspneic and would lie in ventralrecumbency after handling for supportive care. At 96 hrafter admission, the patient was found nonresponsive tostimulus and lying in ventral recumbency with extendedhead and wings, severe dyspnea, and open-mouth breath-ing. The patient subsequently died. One bald eagle (case 3)exhibited generalized weakness and lethargy on admission.Twelve hours after admission, the patient remained weak,resting in sternal recumbency, with moderate dyspnea.Thirty-six hours after admission, the patient remainedweak and was standing with the neck in ventroflexion. Sixtyhours after admission, the patient was found in ventralrecumbency and unable to lift its head, and the eagle diedshortly thereafter. Complete blood cell counts revealedleukocytosis with heterophilia in the 3 hospitalized birdsand lymphocytosis in both hospitalized bald eagles(Table 1).

Whole-body radiographs of the golden eagle (case 1)revealed numerous metallic projectiles in the subcutaneoustissue, near 1 kidney, and embedded in the atlas (C1). The

From the Department of Veterinary Population Medicine/Veterinary Diagnostic Laboratory, College of Veterinary Medi-cine, University of Minnesota, St. Paul, MN (Wunschmann), theDepartment of Pathology, Microbiology and Immunology,University of California, Davis, CA (Rejmanek, Conrad), theWildlife Center of Virginia, Waynesboro, VA (Hall), The RaptorCenter, University of Minnesota, St. Paul, MN (Cruz-Martinez),the Veterinary Associates Stonefield, Louisville, KY (Vaughn),and the California Animal Health and Food Safety LaboratorySystem, University of California, Davis, CA (Barr).

1 Corresponding Author: Arno Wunschmann, Department ofVeterinary Population Medicine, University of Minnesota, 1333Gortner Avenue, St. Paul, MN 55108. wunsc001@umn.edu

J Vet Diagn Invest 22:282–289 (2010)

282

bird had a right ulnar fracture with callus formation andseveral metallic fragments approximately 5 cm distal to theelbow. Radiographic findings in a bald eagle (case 3)included a fracture of the left coracoid immediately lateralto the articulation, with the sternal coracoidal facet and asmall metallic fleck in the soft tissue of the caudoventralabdomen.

The blood-lead level was below the detection level in thegolden eagle. Blood-lead levels were mildly to moderatelyelevated at 0.35 ppm and 0.25 ppm in 2 cases (cases 2 and4), consistent with subclinical cases of lead intoxication,and was greater than 0.65 ppm in the third bald eagle (case3), consistent with a case of lead toxicity.

Necropsy confirmed emaciation in all the animals andconfirmed the presence of the reported fractures in cases 1,3, and 4. In addition, the lungs of 2 bald eagles (cases 2 and3) had areas of dark-red discoloration. The pericardial sacsof these 2 birds contained approximately 2 ml of a clear,amber-colored fluid. Heart, liver, spleen, kidney, brain(cases 1, 2, 4), lung (cases 1–3), and skeletal muscle (cases 1and 4) were collected for histological examination.

Parasite-related histopathological findings were re-stricted to brain, lungs, heart, and skeletal muscle of theeagles. Liver, spleen, and kidney did not have significantlesions in any of the 4 cases. Central nervous system lesionswere present in the golden eagle and a bald eagle (cases 1and 4). The neuropil of the ventral brainstem of the goldeneagle was characterized by the presence of numerous clearspaces (‘‘spongiform state’’) and multifocal glial nodules.Smaller foci of gliosis were also present in the cerebellarcortex of this bird, often extending from the molecularlayer into the granular cell layer. Occasional blood vesselswere surrounded by a few macrophages, lymphocytes, andplasma cells. A mild infiltrate of similar composition waspresent multifocally in the meninges. Multiple profiles ofprotozoal organisms compatible with a Sarcocystis sp. werepresent within both the ventral brainstem and cerebellarlesions, and consisted of schizonts in various stages of

development that ranged from immature schizonts withlarge central round to oval to multilobulated nuclei andperipheral pale amphophilic cytoplasm, to mature schiz-onts that contained clusters of small elliptical merozoites,roughly 1–2 3 4–6 mm, with central nuclei. Merozoiteswithin the mature schizonts were clustered randomly(Fig. 1A) or occasionally arranged in a rosette patternaround central pale residual bodies. The organisms werepresent within neurons (e.g., Purkinje cells) and extracel-lularly in the neuropil. The affected bald eagle (case 4) hada moderate widespread perivascular infiltration, withplasma cells, lymphocytes, and macrophages in cerebrum,mesencephalon, and cerebellum. Numerous microglialnodules were present in the molecular layer of thecerebellum (Fig. 1B). Very infrequent protozoal organismswere found within or adjacent to some of these microglialnodules. The organisms consisted primarily of singleimmature schizonts with large round to lobated basophilicnuclei surrounded by pale amphophilic cytoplasm with rareclusters of merozoites (mature schizonts) or clusters of freemerozoites, the latter seen within foci of inflammatory cellnecrosis. The lungs were the site of significant protozoa-associated findings in cases 2 and 3. The lungs of these baldeagles had random capillary thrombi and intra-endothelialprotozoal schizonts and merozoites. The protozoal schiz-onts were compressed within the pulmonary capillaries,which resulted in elongated serpentine profiles. Some ofthese schizonts were mature and contained numerous well-formed merozoites whereas others were immature and hadlarge basophilic serpentine nuclei (Fig. 1C). The affectedlungs had a mild multifocal lymphoplasmacytic, histiocyticand polymorphonuclear infiltration of the interstitium.

Protozoal tissue cysts without an inflammatory responsewere present in the skeletal muscle of both eagles withencephalitis (cases 1 and 4) and in the hearts of both eagleswith pneumonia (cases 2 and 3). Sections of multipleskeletal muscles, including pectoral muscle, ocular muscle,and longissimus muscle, contained several protozoal tissue

Table 1. Body weight, hematocrit, total protein, and blood cell count in cases 1–3 with reference values for golden eagles (Aquilachrysaetos) and bald eagles (Haliaeetus leucocephalus).*

Golden eagle

(reference values)2,9 {Bald eagle

(reference values)18,20 {Golden eagle,

case 1

Bald eagle

Case 2 Case 3

Body weight (g)

Female 4,900 5,200 2,700 3,200Male 4,100 2,340

Hematocrit (%) 43.3 (65.7) 44 (64) 18 22 32

Total protein (g/dl) 3.3 (60.5) 4 (61) 1.8 2 3

Leukocytes/ml 12,900 (66,282) 12,800 (64,800) 50,400 44,800 34,000

Heterophils/ml 8,295 (64,983) 9,600 (61,664) 47,376 38,870 22,900

Lymphocytes/ml 3,416 (63,683) 2,304 (61,280) 3,024 4,480 11,260

Eosinophiles/ml 892 (6990) 512 (6384) 0 0 0

Monocytes/ml 531 (6611) 384 (6384) 0 450 1,060

* Note that none of these parameters was determined for case 4.{ Numbers in parentheses are standard deviations.

Case Reports 283

cysts in the golden eagle. These cysts were elongated inshape that conformed to the host myofibers and were up to30–40 mm in diameter and 250 mm in length. The tissue-cystwalls were up to 3–4 mm thick and had fine villousprojections (Fig. 1D). Numerous bradyzoites were visiblewithin the cysts, which lacked any visible septation. Rareprotozoal tissue cysts were also present in the skeletalmuscle of the bald eagle (case 4). They ranged from 21 to57 mm 3 65 to 547 mm, with visible internal septa,metrocytes, and closely packed bradyzoites. The tissue-cystwalls ranged from 1 to 4 mm in width, with distinct long,thin, villous-like projections in those cysts with thickerwalls. Tissue cysts and protozoal schizonts were detected inthe hearts of bald eagle cases 2 and 3. There was a mild-to-moderate plasmacytic myocarditis, with rare protozoalzoites and few protozoal tissue cysts in 1 animal (case 2)and a moderate multifocal histiocytic and polymorphonu-clear myocarditis with cardiomyocyte degeneration and

rare large protozoal tissue cysts in the other animal (case 3).The tissue cysts in case 2 ranged from 21 to 72 mm 3 23 to163 mm. The cyst wall ranged from 1 to 3 mm in width andhad distinct fine, thin, villous-like projections. These cystscontained numerous tightly packed bradyzoites withinternal septation and metrocytes along the periphery andangles of the internal septa. Two tissue cysts examined inthe heart of case 3 ranged from 44 to 99 mm 3 124 to209 mm. These cysts had a thin wall approximately 1 mm inwidth that had fine striations suggestive of villousprojections. Bradyzoites were tightly packed within thecysts, which had distinct thin internal septa.

The immunohistochemistry (IHC) methods were identi-cal to those previously described.26 By IHC, merozoites andschizonts detected in the samples of brain (cases 1 and 4),lung (cases 2 and 3), heart (cases 2 and 3), spleen (case 2),and skeletal muscle (case 4) all reacted strongly positivewith polyclonal antiserum raised in rabbits against tissue-

Figure 1. Photomicrograph. A, brain stem, case 1: a cluster of protozoal merozoites is lying freely within the neuropil (arrows).Hematoxylin and eosin (HE) stain. Bar 5 10 mm. B, brain, case 4: a discrete focus of gliosis with mononuclear inflammatory cells ispresent. The focus lies within the molecular layer and extends to the Purkinje cell layer. HE stain. Bar 5 50 mm. C, lung, case 2: a singlemature protozoal schizont composed of a cluster of merozoites is lying within an endothelial cell (arrows). The schizont fills the bloodcapillary and is conforming to the shape of the capillary lumen, resulting in a serpentine shape. At the right is a portion of the lumen of apulmonary atrium. HE stain. Bar 5 10 mm. D, skeletal muscle, case 1: a single relatively thick-walled protozoal tissue cyst containsseveral individual protozoal zoites (metrocytes and/or bradyzoites). HE stain. Bar 5 25 mm.

284 Case Reports

culture–derived whole S. falcatula merozoites (Fig. 2A–C).A small subset of the merozoites in the brain of 1 eagle(case 1) also reacted weakly positive to polyclonalantiserum against S. neurona, whereas small numbers ofmerozoites in the lung (case 3) reacted weakly with themonoclonal antibody raised against S. neurona but did notreact to the polyclonal S. neurona antiserum. The IHCreactivity of the tissue cysts varied. In the golden eagle (case1), the walls of the muscle cysts generally reacted positivewith the polyclonal S. neurona antisera, whereas thebradyzoites reacted weakly positive with the S. falcatulapolyclonal antisera (Fig. 2D, E). In the remaining birds, themajority of tissue cysts did not react to either of theseantisera. None of the merozoites or tissue cysts reacted withpolyclonal antisera to Toxoplasma gondii or Neosporacaninum.

Sections of deparaffinized skeletal muscle from thegolden eagle (case 1) that had the highest density ofmuscle-tissue cysts were processed for transmission electronmicroscopy (TEM) as previously described.25 A total of 3tissue-cyst profiles were located by TEM and measuredapproximately 11 mm 3 23 mm, 18 mm 3 22 mm, and 19 mm3 19 mm. Details of the bradyzoite and metrocytemorphology were obscured because of postmortem tissuedeterioration and the processing of formalin-fixed depar-affinized material. The tissue cysts contained approxi-mately 21–45 individual zoites, which were 2.0–2.3 mm inwidth, with a single zoite that was 4.8 mm in length. Nointernal septa were visible. The cyst walls had long narrowprojecting villous-like structures. The smallest cyst, consid-ered to be an immature cyst, had a wall with villous-likeprojections that ranged from 0.17 to 0.23 mm in width and0.81 to 0.86 mm in length. The remaining 2 cysts, consideredto be more mature, had longer villous-like projections thatwere 0.22–0.40 mm wide and 1.52–2.23-mm long (Fig. 2F).Features common to all of the villous-like projectionsincluded an electron dense 0.03–0.54 mm peripheral layerand long, thin, dense, filamentous structures that extendeddown through the centers of the villous structures thatvaried in number from 1 or 2 filamentous structures in thesmaller, more immature cyst to 6–10 filamentous structuresin the longer villi of the more mature cysts (Fig. 2F). Thesurface of the longer villi of the more mature cysts also hada distinct, regular undulating or ‘‘hobnailed’’ surface(Fig. 2F).

Blocks of paraffin-embedded brain and skeletal muscle(cases 1 and 4), lung and heart muscle (case 2), and lungtissue (case 3) were examined by polymerase chain reaction(PCR) for the presence of protozoal DNA. The methods ofDNA extraction, PCR conditions, and reaction times wereidentical to those previously described.26 Apicomplexanpanspecific primers that target the first internal transcribedspacer (ITS1) region were used to amplify extracted DNAin a nested PCR reaction. The outer (ITS1DF 59-TACCGATTGAGTGTTCCGGTG-39, ITS1DR 59-GCAATTCACATTGCGTTTCGC-39) and inner (ITS1-diF 59-CGTAACAAGGTTTCCGTAGG-39, ITS1diR 59-TTCATCGTTGCGCGAGCCAAG-39) primers were de-signed based on the primers reported in 2 studies (MillerRH, Nandra AK, Stitt T, et al.: Unexpected diversity in

tissue-cyst coccidia [Toxoplasma gondii and Sarcocystisspp.] infecting wild birds in Western Canada. Submitted forpublication).23 The PCR products from positive bands werecleaned by using a commercial PCR clean-up systema andsequenced at the DBS sequencing facility at the Universityof California, Davis, California. A BLAST search was usedto compare the resulting sequences to similar sequencesavailable in GenBank. The PCR analyses of each tissueexamined from cases 1–3 revealed a single (approximately1,000 bp) amplification product that was consistent withthe predicted amplification product size of S. falcatula,whereas no PCR amplification products were detected fromthe brain or muscle tissue of 1 bald eagle (case 4).Subsequent sequencing results from positive bands (cases1–3) were highly similar to S. falcatula and differed from S.neurona. The resulting ITS1 sequences from the brain andskeletal muscle (GenBank accession no. FJ904650) of case1 as well as lung (GenBank accession no. FJ904651) andheart muscle of case 2, were 98% similar to several S.falcatula sequences in GenBank. The sequences were 96%similar to the most closely related S. neurona sequences andonly 92% similar to a Sarcocystis lindsayi ITS1 sequence.The ITS1 sequence from lung of case 3 was 99% similar toseveral S. falcatula sequences, 97% similar to the mostclosely related S. neurona sequences, and only 91% similarto an S. lindsayi sequence. Sequences from all 3 cases werealigned with multiple other Sarcocystis ITS1 sequences andan unweighted pair group method with arithmetic mean(UPGMA) phylogenetic tree was generated by using theprogram ClustalX (version 2.0.8).10 A UPGMA treeshowed that the ITS1 sequences from cases 1–3 clusterwith several other previously described S. falcatula ITS1sequences and are more distantly related to either S.neurona or S. lindsayi (Fig. 3).

The histopathological findings, IHC immunore-activity, and PCR results of the protozoal schizonts andmerozoites in the brain and lungs in cases 1–3 confirm thatthese eagles were infected with a parasite that was highlysimilar and most likely identical to S. falcatula. Thesequencing of the ITS1 region of multiple protozoa of theeagles of this study produced a 98% to 99% match with ITSsequences from S. falcatula in GenBank. Compared withS. neurona, there appears to be more natural sequencevariation among different S. falcatula strains across theITS1 region. Therefore, it is not surprising that theS. falcatula sequences identified in the present study werenot 100% identical to each other or to previouslypublished S. falcatula ITS1 sequences. However, theITS1 region has been shown to differentiate S. falcatulafrom S. neurona.12,23

Of the 4 eagles examined, only the Sarcocystis tissue cystsin the muscle of the golden eagle reacted with the antiseraas previously reported for S. falcatula tissue cysts, whereasthe immunoreactivity of tissue cysts in the other birds wasnegative.6 These results are consistent with previousfindings that indicate that IHC with polyclonal antisera isuseful for the detection of both S. falcatula and S. neuronaschizont and merozoite stages in known susceptible hostspecies. It is also useful for preliminary detection ofS. falcatula, S. neurona, or closely related protozoans in

Case Reports 285

Figure 2. Photomicrograph. A, cerebellum, case 1: strongly immunoreactive protozoal schizonts and/or merozoites in the neuropilare present within the internal granular layer and in the cytoplasm of 1 Purkinje cell. Envision system horseradish peroxidase (HRP):Sarcocystis falcatula polyclonal antiserum. Bar 5 25 mm. B, brain, case 4: the profiles of strongly positive protozoal schizonts and/ormerozoites form a thin string through an area of gliosis with mixed mononuclear inflammatory cell infiltrate within the molecular layer ofthe cerebellar cortex. A portion of the cerebellar cortical internal granular layer is visible at the bottom left of the figure. Envision systemHRP: S. falcatula polyclonal antiserum. Bar 5 50 mm. C, lung, case 2: numerous strongly immunoreactive protozoal schizonts and/orsmaller merozoites are present within capillaries. Envision system HRP: S. falcatula polyclonal antiserum. Bar 5 25 mm. D, skeletal muscle,case 1: metrocytes and/or bradyzoites within a protozoal tissue cyst are immunoreactive. Envision system HRP: S. falcatula polyclonalantiserum. Bar 5 25 mm. E, skeletal muscle, case 1: the wall of the protozoal tissue cyst is immunoreactive. Envision system HRP:

286 Case Reports

rS. neurona polyclonal antiserum. Bar 5 25 mm. F, transmission electron microscope micrograph, skeletal muscle, case 1: one segment of aprotozoal tissue-cyst wall is depicted. The electron lucent inner granular layer of the cyst wall lies at the bottom of the figure. The cyst wallhas numerous thin, outward, villous-like projections with a lucent central region that is bordered by a thick electron dense layer. Althoughthe surface of this layer appears to be relatively smooth, a faint scalloped surface contour is present (arrows). Single, fine, thin, electron-dense filaments extend longitudinally through the center of these projections. At the top of the figure, there is a portion of a striated skeletalmyofiber. Bar 5 0.5 mm.

Figure 3. An unweighted pair group method with arithmetic mean (UPGMA) phylogenetic tree, showing the relationship betweenfirst internal transcribed spacer (ITS1) sequences from eagle cases 1–3 with previously published Sarcocystis falcatula, Sarcocystisneurona, and Sarcocystis lindsayi ITS1 sequences with their respective GenBank accession numbers. The UPGMA tree was created byusing the program ClustalX (version 2.0.8).

Case Reports 287

unknown host species but is neither consistent nor specificfor detection of the tissue-cyst stage of these protozo-ans.6,14,21 The morphologic features support the PCRresults that the muscle of the golden eagle harbored S.falcatula tissue cysts, but the identity of the tissue cysts inthe muscle of the remaining birds was not determined. Inthe golden eagle, there was a relatively large number ofmuscle-tissue cysts that had walls consistent with S.falcatula, based on ultrastructural characteristics such asshape and size of the projections with central filamentousstructures and the peripheral electron dense layer with a‘‘hobnailed’’ contour.4,15 The muscle of the golden eagledid not have contaminating protozoal schizonts based onIHC, so that the amplified gene sequence can be attributedto the tissue cyst.

Although protozoa in the brain of the bald eagle in case 4reacted to S. falcatula antiserum in an identical fashion tothat in cases 1–3, PCR of both brain and muscle werenegative for Sarcocystis spp. This negative PCR result mayreflect the low density of protozoa present in the brain andmuscle of this bald eagle, because the PCR procedure wasdesigned to detect a wide range of Sarcocystis spp.

The presence of replicative schizonts in the tissues ofgolden and bald eagles confirmed that these species areintermediate hosts of S. falcatula. The detection of S.falcatula tissue cysts in the golden eagle suggests thatfalconiformes are true, rather than aberrant intermediatehosts. Before this report, susceptible intermediate hosts ofS. falcatula were thought to be limited to birds within the 4phylogenetic orders: Psittaciformes, Passeriformes, Colum-biformes, and Strigiformes.3,4,6,11,22,24,26

North and South American opossums are recognized asthe definitive host for S. falcatula.3–5,7 Among birds ofprey, unidentified Sarcocystis spp. were previously reportedas a cause of encephalitis in a bald eagle, golden eagle, andnorthern goshawk (Accipiter gentilis).1,8,17

Clinical disease occurs in intermediate hosts of S.falcatula in association with tissue destruction and inflam-mation associated with asexual protozoal multiplication(i.e., schizogony) after an initial oral infection. Among the4 birds described in this study, 2 had proliferation ofmerozoites in pulmonary endothelium and 2 had enceph-alitis, with proliferation of merozoites within neurons andglial cells. Dyspnea and death associated with the ruptureof infected pulmonary endothelial cells is the most commonform of clinical disease seen in psittacines followingingestion of S. falcatula sporozoites, although primaryencephalitis from either rupture and necrosis of brainendothelial cells or within the brain parenchyma also hasbeen reported to occur due to S. falcatula infections.19,24,26

Intermediate hosts, such as the eagles in the present study,may typically become infected with S. falcatula via oralingestion of feed contaminated with opossum feces thatcontained sporocysts. It is also possible that transmissionto the birds described in the current study may haveoccurred through direct ingestion of opossums thatharbored S. falcatula sporocysts within their intestinaltract, because both golden eagle and bald eagle are knownto occasionally prey on opossums.13,16

In conclusion, eagles may serve as intermediate hosts forS. falcatula. This parasite should be considered in thedifferential diagnoses for eagles with chronic disease,neurological disease, and/or rapidly progressive respiratorydisease. Polyclonal antisera are useful diagnostic tools thataid in the detection of S. falcatula or S. falcatula–likeschizonts and merozoites in tissues by IHC. However,confirmation of a S. falcatula infection by PCR, culture, orthe detection of specific ultrastructural features in conjunc-tion with IHC is warranted for unequivocal identificationof S. falcatula.

Acknowledgements. The authors thank Zach Walker ofthe Dwight Chamberlain Rehabilitation Center (HardyLake Reserve) as well as Drs. Mark Ruder and DaveMcRuer for veterinary care and review of the manuscript.The authors are also indebted to the CAHFS histologylaboratory for technical support on IHC, to Bob Nord-hausen for his electron microscopy expertise, and toAmandee Nandra and Michael Grigg for sharing unpub-lished reagents and for their assistance developing thenested primers across the ITS1 region.

Sources and manufacturers

a. ExoSAP-ITH, USB Corp., Cleveland, OH.

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