Sudden Death in Psittacine and Non-Psittacine Birds Associated with Hepatic Infection with an Unclassified Haemosporozoan Parasite: Eight Cases (1994-1996)

D. Greg Hall, Barry G. Harmon, Elizabeth W. Howerth, Christopher R. Gregory, and Susan L. Clubb

Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602-7388 (USA) (Hall, Harmon, Howerth, Gregory) and Parrot Jungle and Gardens, P.O. Box 508, Loxahatchee, FL 33470 (USA) (Clubb)

Abstract. Tissues from eight birds that had died suddenly were submitted over a two-year period (1994 to 1996) to the mail-in histopathology service at The University of Georgia Department of Pathology. Six of the eight birds were psittacines and seven originated from the same area in Florida where the birds had been maintained in screened outdoor aviaries. The birds ranged in age from 2 months to adult. All had been in excellent condition but died suddenly without prior clinical signs, typically in April or May. Submitting veterinarians who performed the necropsies typically reported gross lesions of hepatomegaly, multifocal dark areas on and within the liver, and blood within the body cavity. Microscopic lesions were characterized by numerous hepatic protozoal megaloschizonts, many centered within large areas of acute hemorrhage. Megaloschizonts were identified less frequently and without associated hemorrhage in lung and/or kidney. Protozoal stages have not been identified in peripheral blood cells in birds that died or in surviving clinically normal birds from affected aviaries. The megaloschizonts are morphologically similar to those of some Leucocytozoon species, however the specific identities of the protozoan and its arthropod vector have not been determined. Death associated with this infectious disease usually appears to follow massive hepatic and/or body cavity hemorrhage.

Key Words: Arthropod vector, Body cavity hemorrhage, Hepatic hemorrhage, Leucocytozoon sp. , Liver, Megaloschizont, Sudden death, Unclassified haemosporozoan parasite

Introduction

The Hemosporidia are protozoa in the Suborder Eucoccidiorida, Class Sporozoasida, and Phylum Apicomplexa. ¹ The hemosporidian life cycle involves transmission of sporozoites to animal hosts by bloodsucking arthropods; schizogeny (or merogony) in tissues such as hepatocytes, macrophages, or endothelial cells; and gametogeny in circulating blood cells. Families within the Suborder Hemosporidia include the Sarcocystidae, the Plasmodiidae, the Babesiidae, and the Theileriidae. "Haemosporozoa of Undetermined Taxonomic Status" include a small group of ill-defined protozoa that have been identified sporadically as large tissue cysts in a variety of infected avian species, but for which gametocytes have not been identified in peripheral blood smears. Intermediate arthropod hosts also have not been identified. The purpose of this report is to briefly describe a small series of cases in which sudden deaths in previously healthy psittacine and non-psittacine birds were associated with presence of hepatic protozoal megaloschizonts.

Case Report and Discussion

Multiple tissues from a 26 week-old Sun Conure (Aratinga solstitialis) were submitted in 10% neutral buffered formalin to the mail-in histopathology service at the University of Georgia Department of Pathology. The bird had died suddenly on April 29 with no prior signs of illness and was necropsied by the submitting veterinarian. Body condition was excellent, the liver was large with multifocal large red-black foci, the spleen was markedly enlarged and the lungs were dark red. Tissues were processed routinely for paraffin embedding and 3 micrometer sections stained with hematoxylin and eosin were examined by light microscopy. In the liver there were numerous, large, randomly distributed protozoal cysts associated with areas of parenchymal disruption and hemorrhage (Figure 1). The cysts (megaloschizonts) were round, approximately 250 to 500 micrometers in diameter, and consisted of a peripheral basophilic rim of protoplasm arranged around a fluid-filled central cavity (Figures 2 and 3).

Figure 1. Avian, Liver, H&E stain. Histologic section of liver with scattered intact protozoal megaloschizonts (arrows) and associated hemorrhages.	Figure 2. Avian, Liver, H&E stain. Higher magnification of an intact megaloschizont. The centers contain a small amount of proteinic fluid and there is a thin peripheral rim of organisms.	Figure 3. Avian, Liver, H&E stain. Higher magnification (100X oil immersion) of organisms within an intact megaloschizont. The cyst center is marked by the asterisk. Small coccoid organisms (merozoites) palisade along the margins of the peripheral rim of protoplasm.

The peripheral rim of protoplasm was up to 30 micrometers thick and contained variable numbers of tiny coccoid merozoites approximately 3 micrometers in diameter. The merozoites formed either densely-packed clusters or palisading rows along the margin of protoplasm. Most cysts had no associated leukocyte response, but some were surrounded by infiltrates of lymphocytes, macrophages, and heterophils. Necrotic hepatocytes were occasionally adjacent to cysts or in larger zones within hepatic lobules. A few megaloschizonts were in the renal interstitium without associated hemorrhage or inflammation. Splenomegaly was due primarily to plasmacytosis. A diagnosis of severe protozoal hepatitis with hemorrhage was reported.

Similar hepatic protozoal cysts have been identified in tissues from eight birds submitted to our mail-in histopathology service from 1994 through 1996 (Table 1). All birds but one originated from aviaries in south Florida. Six birds were psittacines, one a passerine (canary), and the eighth a Toucan (order Piciformes). All were in excellent body condition and died suddenly without recognized signs of illness. All but one death occurred in late April or May (the exception occurred in November) and necropsies were performed by the submitting veterinarians. Reported abnormalities included multifocal black foci or suffusive hemorrhages on the liver (5/8 birds), hepatomegaly (4/8 birds), splenomegaly (3/8 birds), and body cavity hemorrhage ("hemoperitoneum") (2/8 birds). In some birds, acute hemorrhages also involved other organs such as heart and intestine but protozoa were not identified in these tissues. In the various cases the megaloschizonts ranged from well-preserved to degenerate, the latter sometimes associated with leukocyte infiltrates. Acute hepatic hemorrhage with or without acute hepatocellular necrosis could be identified around both well preserved (viable) and degenerate cysts. Rare renal (two birds) and pulmonary (one bird) megaloschizonts were also occasionally identified histologically. No parasitized blood cells were identified in tissues from any of these birds. After identification of infection, peripheral blood smears from surviving birds in affected aviaries were examined. No parasitized blood cells were identified in any sample.

In most affected birds, death was attributed to hemorrhage into the liver and/or body cavity around one or more cysts. Reports of similar cases are rare (additional cases in lovebirds are described in another paper submitted to this conference²). An outbreak in April to June of 1982 in Texas killed 65 of 150 canaries. ³ The birds had been housed in a mosquito-proof area with no known exposure to rodents or free-flying birds. Necropsies of nine birds revealed hemorrhage within thoracoabdominal cavities and hepatic lesions with protozoal cysts virtually identical to those described above. No parasitized cells were identified in postmortem blood smears. All assays for bacterial pathogens, viruses, and toxins were negative. The surviving birds were treated with the antiprotozoal drug dimetridazole and no further deaths were reported.

Three apparently healthy canaries obtained during this outbreak were placed with eight canaries in a screened and reportedly mosquito-free aviary. The three introduced birds remained healthy but three resident canaries died suddenly during June of the following year. Two of the three were necropsied and gross and microscopic lesions were identical to those reported for the initial outbreak. Postmortem blood smears were again negative for hemoparasites. Examinations of peripheral blood smears from the three canaries surviving both of these outbreaks were not described in this paper.

The identity of the protozoal pathogen described herein remains undetermined. They are listed by Gardiner, et al. among the "Haemosporozoa of Undetermined Taxonomic Status". ⁴ The megaloschizonts are morphologically similar to those of a variety of Leucocytozoon species including Leucocytozoon simondi in domestic and wild ducks and geese,⁵ L. caulleryi in chickens,⁶ L. dubreuili and L. fringillinarum in robins and grackles. ⁷ All of these Leucocytozoon species are spread to avian hosts by black fly vectors of the genus Simulium. In natural hosts, protozoal sporozoites injected by the fly vector enter the circulation and undergo schizogony in cells such as Kupffer cell macrophages. These small schizonts form merozoites that either infect blood cells and undergo gametogeny, form additional small schizonts in hepatic cells, or form megaloschizonts in many tissues. ⁸ Leucocytozoon simondi megaloschizonts are detectable as early as 4 to 6 days after infection. Schizonts and megaloschizonts eventually rupture to release merozoites that infect blood cells and form gametocytes. Leucocytozoon gametocytes may occur in elongate forms that distort the size and shape of infected blood cells or in round forms that leave infected blood cells undistorted. Gametocytes ingested by the fly vector undergo sporogony to continue the cycle. A specific arthropod vector for the protozoan described here has not been identified, but probably exists and presumably has not been totally excluded from the screened aviaries that housed the infected birds.

Identification of the protozoal parasite described here as a Leukocytozoon or some other species remains to be accomplished. Paraffin-embedded tissue containing the best preserved organisms in our series was reprocessed for and examined by transmission electron microscopy, however the ultrastructural morphology was insufficiently preserved to provide additional useful information as to the specific identification of the parasite (data not shown). Antibodies useful for tissue immunostains and specific for Leukocytozoon tissue stages are not routinely available.

Gametocyte-infected cells have never been identified in blood smears from dead or surviving birds in affected aviaries. There are at least three possible explanations for the failure to identify the gametocyte form of this protozoal pathogen. The parasite may be native to the southern United States and these avian species are unnatural hosts in which gametogeny cannot occur or that die prior to gametogeny. Alternatively, gametocytes may develop but have simply remained undetected in peripheral blood of the dead and/or surviving birds. In the paper describing the outbreak in Texas canaries,³ some sentinel birds housed with putatively exposed birds in a screened facility subsequently died with protozoal hepatic lesions. This could indicate, but does not prove that gametocytes developed in the exposed birds and spread to the naïve birds via an arthropod vector that was not excluded from the screened facility. If so, then the infection was patent and apparently sublethal in the previously exposed birds, which remained healthy for at least a year following the initial outbreak.

Finally, in contrast to the high rate of infection and mortality in the 1982 outbreak in Texas canaries, the incidence of infection in our series of cases appears quite sporadic in both psittacine and non-psittacine species. Many additional pieces of information are needed to more completely understand this infectious disease, including identification of the intermediate host, detailed ultrastructural morphology of the organisms in megaloschizonts, and identification of gametocyte stages in native or non-native species. Participants in this virtual conference who may have encountered similar cases are invited to share their insights and experiences.

Acknowledgments

We wish to thank Dr. Ellis C. Greiner at the Department of Pathobiology, College of Veterinary Medicine, University of Florida for reviewing slides and helpful discussion.

References

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