Five days later, a 32-year-old, female, Southern rockhopper penguin (case 3) from the same institution began to exhibit weakness, anorexia and dyspnea, and auscultation revealed harsh lung sounds. The bird was treated with ponazuril2 (25 mg/kg PO, once), enrofloxacin3 (15 mg/kg SC, once), meloxicam4 (0.5 mg/kg IM, once), and furosemide5 (0.2 mg/kg IM, once) for presumed pulmonary edema, but died 1 h later. Necropsy examination revealed dark red, wet lungs, and an enlarged, nodular spleen. The caudal aspect of the left lung contained a small, focal, tan to brown, firm area.
Cytologic examination of lung tissue imprints from case 3 showed crescent-shaped, 2 x 4-8 mum protozoal zoites (Figure 1B), toxic heterophils and multinucleated giant cells. A complete set of tissues from cases 2 and 3 were fixed in 10% neutral-buffered formalin and submitted to the University of Georgia Zoo and Exotic Animal Pathology Service for histopathology. Fresh spleen from case 2 was submitted to Athens Veterinary Diagnostic Laboratory for aerobic and anaerobic cultures. Fresh lung and liver from Cases 2 and 3 were submitted to the Southeastern Cooperative Wildlife Disease Study (SCWDS, Athens, GA) for molecular testing.
Representative sections of all submitted tissues were routinely processed, embedded in paraffin wax, and 4-5-micron-thick sections were stained with Hematoxylin and Eosin (H&E) for light microscopy. For case 1, the only relevant immunohistochemical stain available at the Connecticut Veterinary Medical Diagnostic Laboratory was a rabbit polyclonal antibody against T. gondii (BioGenex, San Ramon, CA). Additional stains for Sarcocystis sp. (rabbit polyclonal antibody) and T. gondii (rabbit polyclonal antibody) were performed at the California Animal Health & Food Safety Laboratory System, Davis, CA as previously described. For cases 2 and 3, immunohistochemistry was performed at the University of Georgia College of Veterinary Medicine Histology Laboratory using antibodies for S. neurona (rabbit polyclonal antibody, 1:500 dilution for 60 min), Neospora caninum (goat polyclonal antibody6, 1:300 dilution for 30 min) and T. gondii (mouse monoclonal antibody6, 1:1,0000 dilution for 10 min).
The clinical, histologic, immunohistochemical, and molecular findings for three penguins with fatal S. falcatula infection are summarized in Table 1. All penguins had severe, necrotizing and lymphohistiocytic interstitial pneumonia. Parabronchi were flooded with hemorrhage, edema, and fibrin (Figure 1C). Air capillaries were obscured by foci of necrosis with fibrin exudation, and accumulations of heterophils, hemorrhage, and protozoal schizonts (Figure 1D). Air spaces were multifocally lined by hypertrophied epithelial cells (Figure 1E). In cases 2 and 3, schizonts were numerous and elongate, often conforming to the shape of capillaries (Figure 1D, inset), while schizonts were fewer and this classic serpentine morphology of S. falcatula was not observed in case 1. In all cases, schizonts occasionally exhibited a "sunburst" arrangement, in which merozoites radiated around a centralized clearing (Figure 1E, inset).
A full set of tissues, including lung, liver, brain, skeletal muscle, and heart was examined for case 1. No extrapulmonary schizonts or sarcocysts were seen, and additional immunohistochemical stains were not pursued. Cases 2 and 3 exhibited mild lymphohistiocytic myocarditis and myositis and schizonts were observed in multiple tissues in these birds (Table 1). Protozoa stained variably PAS-positive on Periodic acid-Schiff reaction stains, and did not stain with Giemsa. On immunohistochemistry, protozoa exhibited strong immunoreactivity (Figure 1F) for polyclonal S. neurona antibodies and variable immunoreactivity for N. caninum and T. gondii antibodies (Table 1).
Cases 2 and 3 had markedly hypercellular spleens with foci of extramedullary granulopoiesis. There were also coalescing foci of coagulative to lytic necrosis, and case 3 had scattered, intraendothelial schizonts (Figure 2A). Under anaerobic conditions, Clostridium perfringens was cultured from the spleen of case 2. Splenic tissue from case 3 was not available for culture. Low numbers of schizonts were present in foci of splenic necrosis for case 3, so sarcosporidiosis is the most likely explanation for this lesion. However, given that low numbers of gram-positive bacilli in one section of skeletal muscle, septicemia cannot entirely be ruled out in case 3.
All cases had multifocal lymphohistiocytic portal hepatitis and extramedullary granulopoiesis (Figure 2B); cases 2 and 3 had low numbers of schizonts within sinusoids (Figure 2B, right inset). All cases also had moderate numbers of sinusoidal macrophages containing fragments of erythrocytes and intracytoplasmic hemosiderin (Figure 2B, left inset).
In addition to fulminant pulmonary sarcocystosis, case 2 had a small, coelomic fungal granuloma. Aspergillosis is suspected but fungal culture and/or PCR would be required for definitive diagnosis and further testing was not pursued. Case 3 had mild air sacculitis and a focal heterophilic granuloma at the caudal aspect of the left lung lobe. This lesion is believed to be related to a prior aspiration event, given the presence of foreign material and lack of microorganisms on special stains for fungi (Gomori Methenamine Silver), bacteria (modified Brown and Brenn Gram), and acid-fast bacilli (Ziehl-Neelsen).
Formalin-fixed lung tissue from case 3 was trimmed into two, 2-mm-thick pieces and transferred to 2% paraformaldehyde, 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.25. After overnight fixation, tissue was rinsed in 0.1 M phosphate buffer, post-fixed for 1 h with 1% buffered osmium tetroxide (OsO4), then rinsed in deionized water and dehydrated in an ascending ethanol series before infiltration with propylene oxide and Mollenhauer's Epon-Araldite resin mixture. Tissue samples were embedded in fresh resin mixture and allowed to polymerize in a 70 C oven. One-micron-thick sections were stained with Toluidine Blue O to select areas of interest before 60 nm sections were obtained and placed on grids. Grid sections were stained with uranyl acetate and lead citrate before examination with a JEOL JEM 1011 transmission electron microscope at 80 kV. Images were captured with an XR80M wide-angle multi-discipline mid-mount CCD camera7.
Transmission electron microscopy revealed protozoa with ultrastructural features compatible with Sarcocystis spp. (Figure 3). Capillary endothelial cells occasionally contained an intracytoplasmic, serpentine schizont, which conformed to the cell shape. One schizont had a multilobulated nucleus with nuclear spindles arranged along the periphery (Figures 3A,B). Merozoites had a round, central nucleus, an anterior conoid and several tear-shaped, electron-dense micronemes at the posterior end (Figures 3C,D).
For case 1, formalin-fixed, paraffin-embedded (FFPE) lung tissue was submitted to the Wildlife Conservation Society (Bronx, NY) and DNA was extracted using a QIAamp DNA FFPE Tissue Kit8. Extracts were tested by conventional PCR using both pan-apicomplexan and Sarcocystis-specific assays targeting the 18S rRNA and internal transcribed spacer 1 (ITS-1) regions, respectively. A small portion of the 18S rRNA gene was amplified as previously described. A portion of the ITS-1 region was amplified using primer P-ITSF and a degenerate primer (ShortITSR; 5'-GGGATTCARTKGYYGAAA-3') designed based on publicly available Sarcocystis ITS-1 sequences. Amplicons were bi-directionally sequenced commercially, analyzed using Geneious R7 (Auckland, New Zealand) to generate a consensus sequence, trimmed of their primers, and analyzed by BLASTn.
The DNA sequence from the 18S gene was 100% identical to Sarcocystis falcatula in Genbank (MH626537, isolate Lorikeet ID #205850) but also identical to numerous other Sarcocystis isolates in GenBank. The DNA sequence from the ITS-1 region was 100% identical to Sarcocystis falcatula in GenBank (MH626538, isolate Lorikeet ID #205850) and Sarcocystis cf. falcatula in GenBank (AF389339). The next closest match for the ITS-1 region was Sarcocystis speeri and Sarcocystis neurona (98-99% identity).
For cases 2 and 3, DNA was extracted from fresh lung and liver using a commercial DNA extraction kit8 at SCWDS. The genus of the organism was determined by screening tissues with primers that amplify a short portion of the 18S rRNA gene of numerous apicomplexan parasites as previously described. Amplification products were visualized in 2% agarose gels stained with GelRed9. Amplicons were gel-purified using a kit8 and bi-directionally sequenced at the University of Georgia Genomics Facility (Athens, Georgia). Chromatograms were analyzed using Geneious R7 and the consensus sequence was compared to other sequences in GenBank.
To further characterize the Sarcocystis sp. in cases 2 and 3, partial cytochrome b gene was amplified with primers CYTB-F and CYTB-R, the ITS-1 region was amplified with primers ITS-234F19 and ITS-720R19, and the partial surface antigen 2 gene was amplified with primers SAG2-F1 and SAG2-R1 as previously described. Amplicons were purified, sequenced, and analyzed as described above. To rule out the possibility of Plasmodium and/or Haemoproteus spp. infection in cases 2 and 3, a nested PCR targeting the mitochondrial cytochrome b (cytb) gene was conducted as previously described using primary primers HaemNFI and HaemNR3 and nested primers HaemF and HaemR2.
For cases 2 and 3, liver and lung samples from both penguins were positive using the Tg18s58F and Tg18s348R PCR protocol and the resulting sequences (302 bp) were identical to each other and 100% similar to numerous Sarcocystis spp. The cytb gene sequences (580 bp) obtained from liver and spleen of cases 2 and 3 were identical and 100% similar to S. falcatula from captive bare-faced ibis (Phimosus infuscatus) from Brazil (KX265018) and Virginia opossum from California, USA (KP871704). The ITS-1 (287 bp) sequences were identical to each other and contained two polymorphic bases. These sequences were 98-99% similar to numerous S. falcatula strains from Brazil and the U.S.A. with some only differing at those two polymorphic bases. The four SAG2 sequences (402 bp) were identical and were 100% similar to the only S. falcatula (GQ851953) sequence available in GenBank. All four tissues were positive for the rpoB gene. The sequences were identical to each other and 100% similar to one S. falcatula (440 bp, KX265017) strain and >99% similar to several other S. falcatula sequences in GenBank (495 bp, e.g., AY164999).