Joint Pathology Center Veterinary Pathology Services WEDNESDAY SLIDE CONFERENCE 2016-2017 C o n f e r e n c e 24 26 April 2017 R. Keith Harris, DVM, Diplomate ACVP Office of the Dean College of Veterinary Medicine University of Georgia Chris H. Gardiner, Ph.D. Head, JCIDS Capability Document Cell Capability Developments Integration Directorate U.S. Army Medical Department Center and School U.S. Army Health Readiness Center of Excellence Fort Sam Houston, TX CASE I: S354/10 or S370/10 (JPC new parakeets had not been introduced into 4002984). the aviary. Signalment: S354/10: Adult female barred Gross Pathology: At necropsy, multiple parakeet (Bolborhynchus lineola). petechiae were present in the myocardium, S370/10: Adult male budgerigar the pectoral muscles and the gizzard walls of (Melopsittacus undulatus). both animals. Liver and spleen were moderately enlarged. History: In August 2010, sudden deaths of parakeets were noticed in an aviary close to Laboratory results: Nested PCR and Berlin, subsequent DNA sequencing of the Germany. Three barred parakeets (Bol- mitochondrial cytochrome b gene derived borhynchus lineola) and two budgerigars from protozoan megalomeronts was (Melopsittacus undulatus) died within 2-5 performed. Phylogenetic comparison of 479 days after a clinical history of reduced bp of the cytochrome b gene with published activity and food intake. Additionally, one sequences of avian hematozoa found 100% barred parakeet and two juvenile identity with avian malaria parasites budgerigars were clinically affected for (Haemoproteus spp.) of European songbirds. about two weeks but finally recovered. The sequence derived from the barred During the past two years, parakeet was identical to the lineage TURDUS2 of Haemoproteus minutus, a 1 Contributor’s Morphologic Diagnosis: Skeletal muscle (S354/10) / gizzard, smooth muscle (S370/10): Protozoan mega- lomeronts, numerous (variable: with myositis, minimal, lymphocytic and heterophilic and occasionally hemorrhage) Contributor’s Comment: Numerous outbreaks of fatal infections with strikingly similar parasitic structures have been Ventriculus, parakeet. There are numerous protozoal megaloschizonts (small arrows) in the gizzard smooth reported in the past decades in European 2,4,10,13 muscle. There is focal hemosiderin pigment within the aviaries. Psittacines from Australia, koilin layer, suggesting focal ulceration and hemorrhage. New Zealand and South America were (HE, 5X). predominantly affected. No cases of African parrots have been reported so far. Diagnosis parasite previously found in the blood of the in all reported cases was based on common blackbird (Turdus merula) in histopathologic detection of megalomeronts Europe. The sequence from the budgerigar in the heart, skeletal and gizzard muscles was identical with the lineage TUPHI1 of a and, to a lesser extent, in other organs such so far unnamed Haemoproteus sp. as the lung. Most often megalomeronts do previously found in the blood of a song only provoke minimal host response, as in thrush (Turdus philomelos) in Bulgaria. In the cases presented here. Previously, the blood smears of the three only clinically parasites were described as Leucocytozoon affected parakeets, no parasitic structures spp. because of their morphologic features. were detectable. Recent studies also suggest that these cases could have been infections of Besnoitia spp. Histopathologic Description: Skeletal (Sarcocystidae).2 muscle (S 354/10), gizzard, smooth muscle (S 370/10, both tissues with some slide Genetic analysis suggests Haemoproteus variation): Numerous large, often fusiform spp. from European songbirds as causative intact and partially ruptured cyst-like agents in both cases presented here.9 In protozoan structures (size up to 800 μm in Europe, asymptomatic blood infections by diameter) are present in the skeletal muscles Haemoproteus spp. have been regularly and the smooth muscles of the gizzards of observed and are especially prevalent in both parakeets. The cyst-like structures songbirds.12 In the German outbreak, (megalomeronts) have a thick pale psittacine species endemic to South Africa eosinophilic outer wall, are partly and Australia were infected with two compartmented by internal septae, and filled different lineages of Haemoproteus spp. that with myriads of basophilic 1-2 μm, oval are known to infect blackbirds and merozoites. Occasionally, adjoining muscle songthrushes (Turdidae), respectively. These fibers are degenerate or necrotic and results suggest that infection was the result scattered are mild hemorrhages. of previously unknown cross-species Additionally, there are multifocal transmission of Haemoproteus spp. between infiltrations by few lymphocytes, heterophils birds of only distantly related phylogenetic and macrophages, individually also forming orders.7,11 Haemoproteus spp. are closely multinucleate giant cells. 2 related to Plasmodium spp. causing avian The pathogenesis in parrots is unknown. malaria.1 Whether the term avian malaria Presumably, tissue damage caused by should be used for haemosporidian parasites megalomeronts in the conduction system of other than Plasmodium spp. is still subject of the heart may cause death in the affected discussion.8,14 We propose to term the birds. disease in parrots “Haemoproteosis” (G. Valkiunas, personal communication).14 Morphologically, the parasitic structures reported here were strikingly similar to the The Haemoproteus spp. identified are highly reported cases of numerous previous prevalent in the native European songbird outbreaks in Europe. However, further population but normally do not cause harm retrospective, epidemiologic and to their hosts. In contrast, the cases reported experimental studies are needed to assess the here suggest that the same parasites may full range of Haemoproteus spp. or other cause overt disease in invading species such hematozoan parasites involved in this as exotic parrots. These parasites that have disease of parrots. Since blackbirds and adapted to European songbirds may cause songthrushes have been introduced to fatal outbreaks in native psittacines of Australia and New Zealand in the 19th Australia, New Zealand, and South America century, there is a concern that these that are raised in captivity. Blood-sucking parasites already have established in these insects such as biting midges (Culicoides), areas and are potentially affecting the the vectors for Haemoproteus spp. of natural population of parrots. passerine birds in Europe may transmit the parasite from songbirds to parrots.8 Since no JPC Diagnosis: 1. Skeletal muscle gametocytes in parrot blood have been (S354/10)/Proventriculus, smooth muscle found and no blood stages have ever been (S370/10): Protozoan megaloschizonts, described in previous reports, a completely multiple, barred parakeet, Bolborhynchus abortive development has to be assumed. lineola/budgerigar, Melopsittacus undulates. 2. Proventriculus: Proventriculitis, ulcerative, focal, moderate with hemorrhage. Conference Comment: There is significant slide variation in this case as a result of tissue submissions from two different psittacine birds from either the skeletal muscle or the proventriculus. Regardless of the tissue section received, conference participants readily identified numerous apicomplexan protozoal megaloschizonts undergoing various stages of degeneration Ventriculus, parakeet. Haemoproteus minutus within either the skeletal or smooth muscle. megaloschizonts with a thick eosinophilic cell wall, As mentioned by the contributor, septation, and numerous zoites (HE, 100X). (Photo courtesy of: Department of Veterinary Pathology, Haemoproteus spp. are a large group of Freie Universitaet Berlin, Germany parasites that are found primarily in birds, 5 (http://www.vetmed.fu- but can also be seen in turtles and lizards. berlin.de/en/einrichtungen/institute/we12/index.html All species of this parasite are transmitted ). via salivary secretions of hematophagic 3 moderator noted that in this stage, intra- erythrocytic gametocytes are not seen, consistent with the history provided by the contributor. Rather than anemia secondary to erythrocyte parasitism, lesions are associated with megaloschizonts within a variety of cell types, such as the muscular layers of the proventriculus and skeletal muscle, in this case.3,5 The conference moderator also noted that similar to previously reported cases, the mega- loschizonts in this case are large (200-500 um), have compartmentalized internal Ventriculus, parakeet: Base of the ulcer in the septae, and are often associated with ventriculus, with necrosis, Heterophilic inflammation, 5 and heme pigment in the overlying koilin layer. (HE, hemorrhage. Within mature mega- 288X) loschizonts, there are numerous packeted merozoites within structures known as biting midges, hippoboscids (louse flies), or cytomeres. As they mature, megaloschizonts tabanid flies containing infectious rupture and merozoites are released into the sporozoites. blood stream. These merozoites then infect erythrocytes and become gametocytes, ready Within the avian host, the sporozoites enter to be ingested by biting flies to complete the the vascular endothelial cells, most often life cycle of the parasite.3,5,10 In previously within the lung, liver, bone marrow, and reported cases, rupture of megaloschizonts spleen where they undergo schizogony to and the release of merozites caused intense form clusters of schizonts filled with inflammation, hemorrhage, and necrosis5; merozoites. Sexual stages occur