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In order to develop any effective means of prevention or therapy against Neospora caninum infection, all three life cycle stages of this parasite have to be taken into account. In this project, the in vitro culture of N. caninum bradyzoites containing tissue cysts has been developed, thus allowing to the initiation of stage conversion without the use of animal experimentation. This enables researchers to study novel aspects of stage differentiation, to investigate novel diagnostic antigens, and explore the usefulness of potentially interesting drugs and drug targets not only in tachyzoites but also in bradyzoites.
NEOSPORA CANINUM AND NEOSPOROSIS
Neospora caninum (Apicomplexa: Eimeriina: Sarcocystidae) is a Toxoplasma-like parasite that was first identified in 1984 in dogs with encephalomyelitis and myositis. It was later also reported in various species of livestock, especially cattle. In the USA, the EU, and many other countries worldwide, neosporosis is the leading diagnosed cause of abortion in cattle, and thus of major economic impact. One possible route of transmission of N. caninum is through the oral uptake of sporozoite-containing oocysts which are shed in the feces of infected dogs (= definitive host). Sporozoites then enter and traverse the intestinal epithelium, infect macrophages and lymphocytes which leads to dissemination throughout the body, and transform to the rapidly proliferating and diseasecausing tachyzoite stage. Subsequently, the host immune response and possibly other factors trigger stage conversion of the parasite to the slowly proliferating N. caninum bradyzoites. Bradyzoites encapsulate themselves within intracellular cysts surrounded by a solid cyst wall. Oral ingestion of tissue cysts by dogs, e.g. through infected meat, will again lead to infection and subsequent oocyst shedding. Bradyzoites can survive within a latently infected but immuno-competent intermediate host for many years without causing any clinical symptoms. However, reactivation of bradyzoites during immuno-suppression (e.g. during pregnancy) leads to severe neosporosis, namely abortion or birth of weak offspring, in cattle.
DOG-CATTLE- AND TACHYZOITE-BRADYZOITE STAGES
Two different intracellular stages of N. caninum occur in tissues of both final (dog) and intermediate (e.g. cattle) hosts: the actively proliferating and diseasecausing tachyzoite stage has been found in many different tissues and cell types, can be vertically transmitted from mother to the fetus, and accounts responsible for acute disease. The slowly proliferating and tissue cyst-forming bradyzoite stage has so far been found only in the central nervous system and muscle tissue. A third stage, the sporozoite-containing oocyst, is formed within the intestinal tissue of dogs and possibly other canids, is placed into the environment by fecal shedding, and is responsible for horizontal transmission from dogs to other animals. Until recently, in vitro culture of N. caninum was limited to the tachyzoite stage, but now tissue culture procedures have been developed to achieve tachzyoite- to-bradyzoite stage conversion, and thus tissue cyst formation, in vitro.
N. caninum BRADYZOITES REPRESENT HIGHLY RELEVANT TARGETS FOR INTERVENTION
Bradyzoites are of particular epidemiological relevance for two reasons (reviewed in Hemphill et al., 2004; 2006): First, if the infected host becomes partially immuno-compromised, as occurs during pregnancy, bradyzoites are reactivated, which leads to bradyzoiteto- tachyzoite stage conversion, and tachyzoites will infect placental and fetal tissue, resulting in abortion or severely impaired offspring. Secondly, in dogs that consume meat infected with N. caninum bradyzoites, the parasite is likely to undergo sexual reproduction within the intestine. This produces oocysts that are excreted in the faeces and can contaminate soil, water and animal fodder. Thus, strategies to prevent neosporosis must also take into account the importance of bradyzoites. This has earlier led to the development of an artificially immuno-compromised mouse model for the production of N. caninum bradyzoites, with obvious unpleasant side effects for these animals (McGuire et al., 1997; Gondim et al., 2001). However, this laboratory animal model-based method has proven rather unreliable, and requires large numbers of animals.
NITRIC OXIDE-INDUCED IN VITRO TACHYZOITE-TOBRADYZOITE STAGE CONVERSION IN CELL CULTURE
Preliminary experiments to achieve N. caninum stage conversion in cell culture were based on the hypothesis that the parasite would undergo tachyzoite-tobradyzoite stage conversion under stress conditions that lead to a severe inhibition of proliferation, synthesis of tissue cyst wall components, and expression of bradyzoite-specific antigens. We systematically investigated the effects of variations in cell culture conditions for these parameters, including the use of different host cell types. Following a long phase of testing and optimizing conditions, N. caninum tissue cyst formation in vitro was first achieved by treatment of infected murine epidermal keratinocyte (MEK) monolayers with a high dose (70μM) of the nitric oxide donor sodium nitroprusside (SNP)) for up to 8 days (Vonlaufen et al., 2002).
Subsequently, a modified and far more economical procedure was developed which enabled us to produce N. caninum tissue cysts containing bradyzoite stage parasites employing Vero cell cultures (Vonlaufen et al., 2004). Immunofluorescence using antibodies directed against a number of cyst wall antigens showed distinct staining of the cyst periphery, indicating that indeed a cyst wall-like structure was synthesized (Figure 1). This was confirmed by electron microscopy, which clearly demonstrated the formation of a distinct cyst wall in bradyzoite, but not tachyzoite in vitro cultures (Figure 2). Additionally, a protocol was elaborated to purify these bradyzoites from cell cultures, and this has now opened avenues to dissect this important life cycle stage of the parasite at the biochemical and molecular level without the extensive use of animal models.