As demonstrated previously, S. calchasi is capable of causing a severe biphasic central nervous disease in the domestic pigeon . Histologically, a severe lymphohistiocytic and necrotizing encephalitis was found in the late neurological phase of disease. So far, intralesional stages of the parasite had not been confirmed in the pigeon’s brains . Because in tissue sections of cerebral sarcosporidiosis the parasitic load can be low or difficult to detect despite extensive pathological lesions [3, 11], we generated and established an anti-merozoite antiserum against S. calchasi. The antibody reliably detected merozoites, schizonts and tissue cysts including bradyzoites by immunohistochemical analysis. Hereby we demonstrate that S. calchasi stages - although only very few - are present in about half of the brains in both clinical phases of PPE. The presence of S. calchasi DNA could be confirmed by PCR results for the ITS1 region from the cerebrum in all but one pigeon. Together the results indicate that S. calchasi is present in the brains of pigeons with PPE in both disease phases and may suggest a direct involvement of the parasite in the development of the cerebral lesions. However, since no direct association of parasitic stages with histopathological lesions was detected, an unknown immunopathological mechanism may trigger the extensive inflammatory lesions. This notion is underlined by one pigeon infected with 102 sporocysts with a strong cellular immune response but negative results for parasite protein and DNA in the brain. To verify this, further experimental studies are needed since it cannot be ruled out that at the onset of central nervous signs all parasites are effectively eliminated by the immune system.
To further clarify the effect of S. calchasi on the cerebral immune response we established a novel panel of primers to measure the expression level of 11 key immune effector genes and 5 reference genes by RT-qPCR. We characterized an anti-parasite response profile of the host immune system. Most notably, during the schizogonic, first phase of disease the important Th1 cytokines IL-12, IL-18 and IFN-γ as well as IL-15 were significantly down-modulated. During this phase, schizonts were present in various organs, most prominently in the liver, spleen and endothelial cells . In the brains, only very few schizonts were detected in the neuropil without discernible lesions or immune cell infiltrations, although the expression of the major pro-inflammatory cytokines IL-1 and IL-6 and the chemokine IL-8 were significantly up-modulated. This may suggest that similar to other members of the Apicomplexa, S. calchasi is capable of manipulating the IL-12/IL-18/IFN-γ axis to evade the cellular immune response .
Compared to closely related members of the Apicomplexa such as Toxoplasma gondii and Neospora caninum very little is known about the host immune response against Sarcocystis infection. IFN-γ has been shown to be essential for the protection against S. neurona neurological disease in mice . IFN-γ is produced by T-cells, natural killer (NK) cells, monocytes and microglia in the brain . While IFN-γ KO mice show severe neurological disease after experimental infection with S. neurona, SCID mice, which still have functional IFN-γ producing NK cells, only develop disease after treatment with neutralizing anti-IFN-γ antibodies . Furthermore, it has been shown that CD8+ T-cells are critical for the protection from meningoencephalitis in C57BL/6 mice , while a humoral immune response seems to play no major role . There is also first evidence that S. neurona may be capable of interfering with the cytokine signaling of the Th1 immune response. IFN-γ production was reduced in lymphocytes extracted from Equine protozoal meningoencephalitis (EPM) positive horses . Isolated peripheral blood lymphocytes from horses with EPM that were co-cultured with SnSAG1 produced significantly less IFN-γ after 48 h  and the cell-mediated immune responses to SnSAG1 were significantly reduced in horses with EPM . Together with the results of this study it is plausible to assume that Sarcocystis spp. in general may exhibit an immune evasion strategy that disrupts IFN-γ signaling. Since the Th1-biased immune response is of major importance in clearing infection with T. gondii and N. caninum[30, 31], a balanced immune-modulation by the parasite is crucial for survival in its host. Impairment of the IL-12 and IFN-γ expression is therefore one central immune evasion strategy of T. gondii[17, 18]. Furthermore, an impaired synthesis of IL-15 reduces the expression of IFN-γ which enhances the survival of T. gondii. IL-15 also activates CD4+ and CD8+ T-cells, of which CD8+ T cells are stimulated to produce IFN-γ . Interestingly, besides IL-12, IL-18 and IFN-γ, IL-15 was significantly down-modulated in the first phase of S. calchasi infection. This temporary immune suppression may, similar to T. gondii, facilitate S. calchasi to infect host cells and to replicate in the absence of a protective immune response.
In contrast to the first phase, the second neurological phase of disease is associated with a massive mononuclear cell infiltration and characterized by a markedly up-modulated IFN-γ expression. Notably, IL-12 and IL-18 as important inducers of IFN-γ were not up-modulated. IFN-γ is the key cytokine for the activation of mononuclear cells. This correlates well with the prominence of T-cells, the granulomatous character of the lesions and prominent MHC-II signaling in the pigeon brains. Chicken IFN-γ increases the expression of class II MHC on antigen presenting cells and directly activates macrophages and natural killer cells which can excrete further inflammatory effector molecules such as TNF-α that may destroy tissue. [34–36]. Since TNF-α has not been identified in the avian genome, we tested TL1A and LITAF which both were significantly up-modulated. Taken together this suggests an extensive Th1-biased T-cell driven immune response, which appears inappropriate in the light of a very low or absent parasite load.
Pigeons infected with S. calchasi depict neurological signs such as incoordination about eight weeks after infection when tissue cysts mainly located in skeletal muscles contain infectious bradyzoites . This alteration in intermediate host behavior may lead to an increased predation rate by the final host, the Northern goshawk. In contrast to pigeons, several natural and aberrant hosts of avian Sarcocystis spp. (i.e. S. neurona) depict neurological signs associated with encephalitis only during the schizogonic phase which does not allow transmission to a final host [7, 9, 37, 38]. Changes in intermediate host behavior as reported for common voles (Microtus arvalis) and lemmings (Dicrostonyx richardsoni) infected by birds of prey-transmitted Sarcocystis cernae and Sarcocystis rauschorum, respectively, have been suggested to enhance parasite transmission due to increased predation rates [39, 40]. However, whether the change in behavior of pigeons induced by S. calchasi may be regarded as a parasite’s adaptation to enhance its fitness or simply as side effect due to a delayed-type hypersensitivity reaction requires further investigation to meet the manipulation hypothesis .
In conclusion, the observations of this study suggest that the Th-1 immune response during the schizogonic phase of the S. calchasi development is down-modulated in the intermediate host. The absence of a strong host’s cellular immune response in the pigeon may facilitate parasite evasion during acute disease and subsequent formation of tissue cysts. The results of this study further suggest that during the late central nervous phase of PPE a T-cell mediated delayed-type hypersensitivity reaction may cause the cerebral lesions.