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Sarcocystis calchasi is an apicomplexan parasite that has been described as the cause of Pigeon Protozoal Encephalitis (PPE) of pigeons in 2009. This central nervous disease of domestic pigeons which is characterized by a biphasic clinical course has so far been identified in Germany and the USA. Until now the pathophysiology of PPE has been unclear. The present work aimed at investigating the immunological response of the domestic pigeons towards an infection with S. calchasi and its ability to interfere with the immune response in PPE. For this reason the brains of pigeons from both clinical phases were examined for the presence of parasite DNA by nested-PCR, which found all but one pigeon positive in both disease phases. Furthermore, a specific antibody for S. calchasi was developed to immunohistochemically analyze the pigeons brains for the presence of parasite protein. During the first phase of disease no cellular immunological reaction was discernable. However, four of eight pigeons had a very low load of positive labeled schizonts in the neuropil. In contrast, all pigeons that had to be euthanized because of severe neurological signs during the second phase of disease had a massive lymphohistocytic immune cell infiltration in the brain.
Importantly, in only three of seven pigeons few tissue cysts of the parasite could be immunohistochemically labeled in unaffected areas.
In the second part of this thesis, the expression of important cytokines in the pigeons brains during both disease phases was examined by quantitative real-time PCR (RT-qPCR). In particular the expression rates of interleukin (IL) 1, IL-6, IL-7, IL-12, IL-15, IL-18, interferon gamma (IFN-γ), TNF-like factor 1A (TL1A), lipopolysaccharid-inducing TNF-alpha factor (LITAF), transforming growth factor β2 (TGF-β2) and of the chemokine IL-8 were measured. For relative quantification of gene expression suitable reference genes had to be chosen and established for RT-qPCR analysis. Therefore, ten reference genes, namely β-actin (betaactin), GAPDH (glycerinaldehyd-3-phosphat-dehydrogenase), Gusb (glucuronidase, beta), HMBS (hydroxymethylbilan-synthase), HPRT (hypoxanthin-hosphoribosyl-transferase), RPL13 (ribosomal protein L13), RPL19 (ribosomal protein L19), RPS7 (ribosomal Protein S7), TFRC (transferrin-receptor) and Ywhaz (tyrosin 3-monooxygenase / tryptophan 5-monooxygenase activating protein, zeta-polypeptid) were established de novo for the pigeon. In addition, the composition of inflammatory cells in the brain lesions of the second disease phase was investigated by specific antibodies. The results show that during the first disease phase (schizogony of the parasite) the cytokines IL-15, IL- 18 and IFN-γ (all of them significantly) and IL-12 were down-modulated when compared with the control group. Proinflammatory cytokines IL-1β, IL-6 and the chemokin IL-8 were up-modulated.
In contrast, in the central nervous, second phase of disease, IFN-γ together with TL1A and LITAF were significantly up-modulated, which correlated with a prominent MHC-II protein expression in areas of mononuclear cell infiltration and necrosis. Immunohistochemically, the mononuclear cell fraction was identified as mainly Tlymphocytes. In conclusion, the results of the present study suggest that S. calchasi may be capable of manipulating the TH1 immune response of the intermediate host during the first phase of disease and by this hinder a protective anti-parasitic cellular immune response. The results further suggest that during the late central nervous phase a T-cell mediated delayed-type hypersensitivity reaction may cause the severe inflammatory cerebral lesions in the pigeons. In combination, it seems plausible that this complex pathology may aid in the spreed of the parasite from the intermediate host to the definitive host.