Fatal toxoplasmosis in Little Penguins (Eudyptula minor) from Penguin Island, Western Australia

Abstract

Routine post mortems of deceased penguins from Penguin Island, Western Australia, found that a temporal cluster of cases presented with characteristic gross and microscopic changes, namely birds in good body condition with hepatomegaly and splenomegaly, multifocal hepatic and splenic necrosis and numerous, 1-2 μm diameter protozoan parasites within the necrotic foci. Electron microscopy identified the protozoa as belonging to the phylum Apicomplexa. Molecular investigations by PCR gave inconsistent results. PCR performed by an external laboratory identified a novel Haemoproteus spp. organism in samples from 4 of 10 cases from this group, while PCR at Murdoch University identified Toxoplasma gondii in 12 of 13 cases (including 9 of the 10 assayed at the external laboratory). Immunohistochemistry of formalin fixed tissues also identified Toxoplasma in the hepatic and splenic lesions. The distinctive mortalities which were observed in this group of penguins are attributed to a fulminant toxoplasmosis, with a concurrent Haemoproteus infection in some cases. Though the clinical signs of infection are unknown, the gross and microscopic appearance at post mortem is sufficiently characteristic to allow a diagnosis to be made on these features. Definitive confirmation of Toxoplasma infection can be made by immunohistochemistry or PCR.

Keywords: Apicomplexa; Haemoproteus; Hepatitis; Little penguin; Splenitis; Toxoplasma.

Graphical abstract

Fig. 1

Enlarged liver with multiple, pinpoint cream to white spots.

Fig. 2

Enlarged spleen with speckled tan areas of discolouration.

Fig. 3

Liver, necrotic focus (the area of relative pallor, within which numerous organisms were identifiable) (5 μm section, Haematoxylin and Eosin stain, 100x).

Fig. 4

Liver, intact and necrotic hepatocytes and numerous protozoa (arrows), free and within cysts (5 μm section, Martius Scarlet Blue stain, 400x). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Splenic impression smear, erythrocytes (E), splenic stromal cells (S) and numerous protozoa (arrows) (Wright's Giemsa stain, 1000x).

Fig. 6

(And detail) – four parasites in a cyst within the cytoplasm of a host cell, spleen (x3810, bar = 2 μm) Image left: E - erythrocyte, Ph - phagocyte, P - protozoa; image right (detail): N - nucleus.

Fig. 7

Protozoan with apical complex (x15000, bar = 1 μm).

Fig. 8

Dividing protozoan forming merozoites (x15000, bar = 1 μm).

Fig. 9

Similarity of Toxoplasma gondii from Little Penguins with representative strains from Archetypals I, II, and III in the B1 gene. A: shows polymorphisms at the 366 nucleotide. B: shows polymorphisms at the 504 nucleotide.

Fig. 10

Liver, several foci of brown staining indicate Toxoplasma antigen within a necrotic focus (x100, Toxoplasma polyclonal antibody IHC). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 11

Liver, staining of protozoan antigen in intact and necrotic hepatocytes (x400 Toxoplasma polyclonal antibody IHC).