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An Investigation of an Infection with a Protozoan Parasite Causing Mortalities in Little Penguins (Eudyptula Minor) on Penguin Island, Western Australia

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An Investigation of an Infection with a Protozoan Parasite Causing Mortalities in Little Penguins (Eudyptula Minor) on Penguin Island, Western Australia An investigation of an infection with a protozoan parasite causing mortalities in Little Penguins (Eudyptula minor) on Penguin Island, Western Australia Kamahl Campbell, BA BSc BVMS (Hons) This thesis is presented for the degree of Master of Science of Murdoch University, 2015. I declare that this thesis is my own account of my research and contains as its main content work that has not previously been submitted for a degree at any tertiary education institution. .................................... Kamahl Campbell Abstract Since at least 2001, routine post mortems of deceased penguins from Penguin Island, Western Australia, have been conducted at Murdoch University Veterinary Hospital (MUVH). In late 2011 and early 2012, a cluster of 12 cases presented with similar and characteristic gross and microscopic changes, namely birds in good body condition with hepatomegaly and splenomegaly, multifocal hepatic and splenic necrosis and numerous, small, 1-2µm diameter protozoan parasites within the necrotic foci. A review of earlier reports in the MUVH archive identified isolated similar cases from 2006 and 2008, which had been provisionally diagnosed as Avian Malaria, that is, Plasmodium spp. infection. An investigation was established in order to: a) definitively identify the parasite causing the mortalities, with the additional aims of: b) evaluating the live population of Penguin Island Little Penguins for the presence of parasitaemia, and c) investigating the possibility that another bird species present on Penguin Island might represent a reservoir of infection. Ninety-four blood smears were made from 79 individual Little Penguins collected from winter to summer of 2012 and in the early spring of 2013. One smear identified intraerythrocytic organisms consistent with the blood stages of an apicomplexan parasite, for example, merozoites or early gametocytes of Haemoproteus or Plasmodium, or merozoites or sporozoites of Babesia. Fifty-one blood smears were made from 51 Bridled Terns (Sterna anaethetus) captured during two visits to the island in November 2012 and March 2013, with no parasites detected in these smears. Electron microscopy of the protozoan parasite identified it as belonging to the phylum Apicomplexa. Further identification to the level of genus was not possible. Molecular identification of the parasite using Polymerase Chain Reaction (PCR) methodology gave inconsistent results. PCR performed by an independent laboratory identified a novel Haemoproteus spp. organism in 4 of 10 cases from this group; however, these results could not be replicated in our laboratory. Additional PCR using a variety of primers aimed at detecting members of the Apicomplexa identified a parasite from the family Sarcocystidae, which was subsequently identified as Toxoplasma. Immunohistochemistry of formalin fixed tissues also identified Toxoplasma in the hepatic and splenic lesions. 1 The distinctive mortalities which were observed in this group of penguins, and which have occurred sporadically since, appear to be attributable to a fulminant toxoplasmosis, with or without a concurrent haemoproteosis in some cases. The significance of the apparent polyparasitism in some of the birds is unknown, as the relative contribution of concurrent Haemoproteus infection to the lesion aetiopathogenesis cannot be quantified at this time. 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 infection may be made by immunohistochemistry or PCR. 2 Table of Contents An investigation of an infection with a protozoan parasite causing mortalities in Little Penguins (Eudyptula minor) on Penguin Island, Western Australia .......................................... 0 Abstract ......................................................................................................................................................... 1 Acknowledgments ...................................................................................................................................... 5 1. Literature review ................................................................................................................................... 7 Preamble .................................................................................................................................................................................7 Little Penguins ......................................................................................................................................................................7 Penguin Island and threats to the population ........................................................................................................8 Haemoparasitic diseases of birds .............................................................................................................................. 13 Haemoparasitic diseases of penguins...................................................................................................................... 15 Other protozoan parasites infecting penguins .................................................................................................... 17 Summary .............................................................................................................................................................................. 17 2. Post mortem examinations ............................................................................................................... 19 Introduction ....................................................................................................................................................................... 19 Materials and methods .................................................................................................................................................. 20 Routine penguin necropsy procedure ............................................................................................................. 20 Results ................................................................................................................................................................................... 21 Case1: 06/1172 ........................................................................................................................................................... 22 Case 2: 08/1075 .......................................................................................................................................................... 22 Case 3: 11/484 ............................................................................................................................................................ 23 Case 4: 11/624 ............................................................................................................................................................ 24 Case 5: 11/626 ............................................................................................................................................................ 25 Cases 6 and 7: 11/627 and 11/628 ................................................................................................................... 26 Case 8:12/020 ............................................................................................................................................................. 26 Case 9: 12/021 ............................................................................................................................................................ 27 Case 10: 12/338 .......................................................................................................................................................... 28 Case 11: 12/362 .......................................................................................................................................................... 29 Case 12: 12/375 .......................................................................................................................................................... 29 Case 13: 12/385 .......................................................................................................................................................... 32 Case 14: 12/473 .......................................................................................................................................................... 32 Case 15: 12/517 .......................................................................................................................................................... 33 Case 16: 12/524 .......................................................................................................................................................... 34 Case 17: 13/081 .......................................................................................................................................................... 35 3 Case 18: 13/670 .......................................................................................................................................................... 36 Other ................................................................................................................................................................................. 36 Summary .............................................................................................................................................................................
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