For many years hexamitids, Hexamita spp. and Spironucleus spp., have frequently been reported in vertebrates, particularly in fish. This suggests a potentially important role of these parasites in the fish culture industry. Though the majority of hexamitids are not known to cause disease in their vertebrate host, those that have been documented as associated with disease are still in need of further investigation into their geographical distribution, host range, life cycle, host-parasite relationship, pathogenicity, diagnosis, prevention, treatment, and control. Spironucleus vortens is a hexamitid recently described from angelfish (Pterophyllum scalare). Although the structure of this parasite has been investigated using the electron microscope (Poynton et al., 1995), other information on this organism is poorly understood. Thus, the purpose of this research was to study the nature of S. vortens in TYI-S-33 culture medium and in the angelfish host. The optimal environmental conditions for S. vortens growth were investigated using variations of temperature, pH, and bile concentrations. This study is useful in helping to understand the locations and environmental conditions in the host that are suitable for the growth of S. vortens. Treatment of S. vortens, using seven chemotherapeutic agents; dimetridazole, metronidazole, pyrimethamine, albendazole, fenbendazole, mebendazole, and magnesium sulfate was evaluated. The pathogenicity of S. vortens in angelfish was investigated in fish experimentally inoculated with trophozoites. This study provided information to help understand the pathogenesis of the parasites in the host. Finally, to examine the protective defense mechanisms, the presence of anti-S. vortens antibodies in angelfish serum were evaluated along with the presence of immune cells (lymphocytes, macrophages, eosinophilic granular cells, neutrophils, and plasma cells) at invaded sites of the intestine and other internal organs in response to an experimental Spironucleus vortens infection. The results of this research provide information on this parasite's effect on the fish host which may be useful in understanding the nature of other hexamitids. A few published reports have suggested the in vitro growth requirement of fish Spironucleus (Poynton et al., 1995; Sterud, 1998), but none have examined the optimal conditions required for growth and the pathogenicity of S. vortens. The first study was to examine the optimal requirements for the in vitro growth of the parasite. The organisms were cultivated in either an artificial medium (TYI-S-33) at different temperatures or various pH conditions, or in medium supplemented with different bile concentrations at 25°C. Criteria used to justify the optimal conditions were average cell number ml-1, growth rate, survival time, and cell conditions (motility and morphology). The organisms survived longest at 22°C, and had the highest average cell number ml-1 at 25°, 28° and 31°C. At 25°C the parasites were highly active and survived up to 6 days. The organisms cultivated at pH 6.5, 7.0 and 7.5 yielded the highest average cell number ml-1 with survival periods up to 13-14 days. Most of the organisms cultivated at a pH lower than 6.0 or a pH higher than 7.5 were suppressed and killed within 5-6 days of cultivation. All cultures supplemented with bovine or fish bile yielded lower maximal numbers of parasites than cultures with no bile. These results indicate that the optimal condition for the in vitro cultivation of S. vortens is 25°C and pH 6.5 to 7.5 without supplementation with bile. In order to treat spironucleosis, the efficacy of various chemotherapeutic agents on the growth of S. vortens was examined in vitro. In this study nitroimidazoles and benzimidazoles, formerly reported as drugs of choice for the treatment of diplomonads, pyrimethamine and magnesium sulfate (Epsom salt) were evaluated at different concentrations on the growth of S. vortens. Dimetridazole and metronidazole were effective in inhibiting the parasite's growth at concentrations of 1 μg ml-1 or higher. Albendazole and fenbendazole suppressed the growth of parasites at concentrations of 1.0 μg ml-1 or higher after 24 h exposure. Mebendazole was the most effective agent of the benzimidazole group; and inhibited the parasite's growth at concentrations of 0.5 μg ml-1 or higher. Pyrimethamine at concentrations of 1-10 μg ml-1 failed to inhibit the parasite's growth. Magnesium sulfate inhibited the growth of the parasites only at high concentrations (70 mg ml-1 or higher) . This study indicates that dimetridazole, metronidazole and mebendazole are the most effective chemotherapeutic agents in vitro at inhibiting the growth of S. vortens. To investigate the pathogenesis of spironucleosis, angelfish were orally (PO) or intraperitoneally (IP) inoculated with S. vortens. Control angelfish which were orally gavaged or intraperitoneally injected with PBS were in normal body condition and had no morbidity or mortality. Compared to the control angelfish, PO-infected angelfish were inappetent with no other clinical signs, while IP-infected angelfish showed clinical signs of inappetite, weakness, respiratory distress, and laying on their sides. Twenty percent of the IP-infected angelfish died within the first three weeks after infection. In PO-infected angelfish, the organisms were located only in the intestinal lumen. In IP-infected angelfish, S. vortens were found in the blood, stomach, intestine, and other internal organs (spleen, gall bladder, and ovary). However, no parasites were observed within the intestinal mucosa of either PO- or IP-infected fish. Histopathologic examination of the intestines revealed mild to moderate multifocal enteritis in both PO- and IP-infected angelfish. The mucosal epithelium appeared undamaged although the parasite was closely located and appeared attached to the intestinal mucosa. The results suggest that S. vortens normally causes mild to moderate multifocal enteritis with no morbidity. However, the parasites can cause granulomatous inflammation in a wide variety of host tissues, and may be lethal if they enter the abdominal cavity and disseminate to other organs via the blood circulation. Immunity, both cell mediated and humoral, against S. vortens was investigated in this study. Histopathologic examination revealed a response from inflammatory cells infiltrated and localized in the affected tissues. Macrophages, lymphocytes, and plasma cells were the most common cell types found in the internal organs. Macrophages were active in the affected tissues where the parasites lived in situ. However, in vitro studies indicated that there were no differences in a production of H₂O₂ or in phagocytosis between macrophages of control and infected angelfish regardless of inoculum dosage and administration route. A preliminary study of humoral antibody indicated that angelfish did not develop anti-S. vortens antibody after they were orally or intraperitoneally infected with either a low or a high number of the organisms. It is suggested that localized leucocyte response may be an important mechanism against Spironucleus vortens infection in angelfish. This research has indicated some of the important environmental factors affecting the parasite's growth, and has provided some initial information on the pathogenicity of S. vortens. In addition, preliminary information on the host's protective immune systems, humoral and cell-mediated immunity, against the parasite have been documented. The results from this research will be useful for aquaculture, particularly of tropical freshwater angelfish, and may help to provide an understanding of the biological roles of other hexamitids. / Ph. D.

The diplomonads are a diverse group of eukaryotic flagellates found in microaerophilic and anaerobic environments. The most studied diplomonad is the intestinal parasite Giardia intestinalis, which infects a variety of mammals and cause diarrheal disease. Less is known about Spironucleus salmonicida, a parasite of salmonid fish, known to cause systemic infections with high mortality. We created a transfection system for S. salmonicida to study cellular functions and virulence in detail (Paper I). The system was applied to explore the mitochondrion-related organelle (MRO) in S. salmonicida. We showed that S. salmonicida possesses a hydrogenosome (Paper II) with a higher metabolic capacity than the corresponding MRO of Giardia, the mitosome. Evolutionary analysis of key hydrogenosomal proteins showed ancient origin, indicating their presence in the ancestral diplomonad and subsequent loss in Giardia. Annexins are of evolutionary interest since these proteins are found across all kingdoms. Annexin-like proteins are intriguingly expanded into multigene families in Giardia and Spironucleus. The annexins of S. salmonicida were characterized (Paper III) with distinct localizations to various cellular structures, including a putative adhesion structure anterior in the cell. The disease-causing Giardia trophozoites differentiate into infectious cysts, a process essential for transmission and virulence of the parasite. Cysts are often spread via contaminated water and exposed to environmental stressors, such as UV irradiation. We studied the survival and transcriptional response to this stress factor (Paper IV) and results showed the importance of active DNA replication machinery for parasite survival after DNA damage. In addition, we studied transcriptional changes along the trajectory of encystation (Paper V), which revealed a coordinated cascade of gene regulation. This was observed for the entire transcriptome as well as putative regulators. Large transcriptional changes appeared late in the process with the majority of differentially regulated genes encoding hypothetical proteins. We studied the localizations of several of these to gain information of their possible function. To conclude, the diplomonads are complex eukaryotic microbes with cellular processes adjusted to match their life styles. The work in this thesis has provided insight of their adaptations, differences and similarities, but also new interesting leads for future studies of diplomonad biology and virulence.

As sequencing technologies advance genome studies are becoming a basic tool for studying an organism, and with more genomes available comparative genomics is maturing into a powerful tool for biological research. This thesis demonstrates the strength of a comparative genomics approach on a group of understudied eukaryotes, the diplomonads. Diplomonads are a group of single cell eukaryotic flagellates living in oxygen-poor environments. Most diplomonads are intestinal parasites, like the well-studied human parasite Giardia intestinalis. There are seven different G. intestinalis assemblages (genotypes) affecting different hosts, and it’s under debate whether these are one species. A genome-wide study of three G. intestinalis genomes from different assemblages reveals little inter-assemblage sexual recombination, supporting that the different G. intestinalis assemblages are genetically isolated and thus different species. A genomic comparison between the fish parasite S. salmonicida and G. intestinalis reveals genetic differences reflecting differences in their parasitic lifestyles. There is a tighter transcriptional regulation and a larger metabolic reservoir in S. salmonicida, likely adaptations to the fluctuating environments it encounters during its systemic infection compared to G. intestinalis which is a strict intestinal parasite. The S. salmonicida genome analysis also discovers genes involved in energy metabolism. Some of these are experimentally shown to localize to mitochondrion-related organelles in S. salmonicida, indicating that they possess energy-producing organelles that should be classified as hydrogenosomes, as opposed to the mitosomes in G. intestinalis. A transcriptome analysis of the free-living Trepomonas is compared with genomic data from the two parasitic diplomonads. The majority of the genes associated with a free-living lifestyle, like phagocytosis and a larger metabolic capacity, are of prokaryotic origin. This suggests that the ancestor of the free-living diplomonad was likely host-associated and that the free-living lifestyle is a secondary adaptation acquired through horizontal gene transfers.  In conclusion, this thesis uses different comparative genomics approaches to broaden the knowledge on diplomonad diversity and to provide more insight into how the lifestyle differences are reflected on the genetic level. The bioinformatics pipelines and expertise gained in these studies will be useful in other projects in diplomonads and other organismal groups.

The diplomonads are a diverse group of eukaryotic microbes found in oxygen limited environments such as the intestine of animals were they may cause severe disease. Among them, the prominent human parasite Giardia intestinalis non-invasively colonizes the small intestine of humans and animals where it induces the gastrointestinal disease giardiasis. Two of the eight genetic groups of G. intestinalis, assemblage A and B, are known to infect humans and have zoonotic potential. At the start of project, genome scale data from assemblage B-H was either sparse or entirely missing. In this thesis, genome sequencing was performed on the assemblage B isolate GS (Paper I) and the P15 isolate (Paper III) of the hoofed-animals specific assemblage E to investigate the underlying components of phenotypic diversity in Giardia. Comparisons to assemblage A isolate WB revealed large genomic differences; entirely different repertoires of surface antigens, genome rearrangements and isolate specific coding sequences of potential bacterial origin. We established that genomic differences are also manifested at the transcriptome level (Paper VIII). In a follow up analysis (Paper IV) we concluded that the Giardia assemblages are largely reproductively isolated. The large genomic differences observed between Giardia isolates can explain the phenotypic diversity of giardiasis. The adaptation of diplomonads was further studied in Spironucleus barkhanus (Paper II), a fish commensal of grayling, that is closely related to the fish pathogen Spironucleus salmonicida, causative agent of systemic spironucleosis in salmonid fish. We identified substantial genomic differences in the form of divergent genome size, primary sequence divergence and evidence of allelic sequence heterozygosity, a feature not seen in S. salmonicida. We devised a transfection system for S. salmonicida (Paper VI) and applied it to the study of the mitochondrial remnant organelle (Paper VII). Our analyses showed that S. salmonicida harbor a hydrogenosome, an organelle with more metabolic capabilities than the mitosome of Giardia. Phylogenetic reconstructions of key hydrogenosomal enzymes showed an ancient origin, indicating a common origin to the hydrogenosome in parabasilids and diplomonads. In conclusion, the thesis has provided important insights into the adaptation of diplomonads in the present and the distant past, revealing hidden diversity.

Durch Parasiten stellen eine starke Gefährdung für die Aquakultur dar. Die durch diplomonaden Flagellaten bei der Regenbogenforelle Oncorhynchus mykiss verursachte Morbidität und Mortalität wurde bisher in Deutschland noch nicht gründlich untersucht. Ich habe diese Parasiten mittels SEM & TEM charakterisiert und wurde die Art Spironucleus salmonis bestimmt. Erstmals konnten die caudale Projektion, sich entleerende Vakuolen und verformbare Kernloben nachgewiesen werden. Ich untersuchte Mikrohabitatpräferenz von des Parasiten sowie pH-Profile in vier Darmabschnitten der Fische. Das Vorkommen und die Dichte von S. salmonis war in der Pylorusregion wesentlich höher als in anderen Bereichen. Das pH-Profil war bei infizierten und nicht infizierten Fischen gleich. Der optimale pH-Wert für S. salmonis war 7,1-7,5. Ich habe die Lebenszyklus der Diplomonaden mittels LM & SEM unter Kulturbedingungen untersucht. Die Enzystierung begann mit der Anheftung von Trophozoiten mit der Spitze der adhäsiven hinteren Flagellen aneinander oder an Fremdkörper. Die pyriformen Trophozoiten wurden kugelförmig, und die vorderen Flagellen inaktiv. Oberflächenbläschen produzierten eine lichtbrechende Zystenwand. Dies ist der erste Beschreibung der Multifunktionalität von Flagellen bei Diplomonaden. Ich untersuchte pathogene Mechanismen und sezierte die Pylorusregion sowie die Leber mittels H&E, PAS/AB. Bei infizierten Fischen trat eine signifikante Hypertrophie der Becherzellen auf. Zu erkennen war eine Hyperaktivität der Becherzellen, jedoch keine Hyperplasie. Ich entwickelte einen in vitro  Plasma-Inkubationstest zur Bestimmung der Suszeptibilität von Regenbogenforelle, Karpfen und Störe. Die unterschiedliche Resistenz von Stör, Karpfen und Regenbogenforelle gegen S. salmonis entsprechend zu den epizootiologischen Daten. Meine Untersuchungen führten zu einem neuen diagnostischen Hilfsmittel, Vorschlägen für neue Behandlungsmethoden, zu verbesserten in vitro-Kulturbedingungen und einem Modellsystem für die Multifunktionalität von Flagellen und flagellaren Signaltransduktion. / Parasitic diseases pose a significant threat to aquaculture. Diplomonad flagellates in rainbow trout Oncorhynchus mykiss are associated with morbidity and mortality; but in Germany has not been thoroughly studied. I characterised the species by SEM & TEM, which revealed Spironucleus salmonis, allowed its complete description including newly showing the caudal projection, discharging vacuoles, and deformable nuclear lobes; diagnostic keys were improved. The microhabitat preference of diplomonads was tested by recording occurrence and density of infection, and pH profile in 4 intestinal regions in fish. Occurrence and density of S. salmonis were significant higher in the pyloric region than elsewhere. The pH profile in uninfected and infected fish was similar; a causal relationship between microhabitat preference and pH was unlikely, and the optimal pH was between 7.1 – 7.5. I described life cycle and encystment using light and SEM. Encystment in culture began by trophozoites attaching at tip of adhesive posterior flagella to each other/debris. Pyriform trophozoites became sub-spherical, anterior flagella inactive, surface blebs produced a refractile cyst wall. Cysts clusters may exceed minimum infective dose for new infection; suggesting new treatment target. This is the first report of multi-functionality of flagella in diplomonads. I investigated pathogenic mechanism of diplomonads by sectioning and staining the pyloric region of the intestine and liver with H&E, and PAS/AB. There was significant hypertrophy of goblet cells in infected fish. The hyperactivity of goblet cells was seen, but no hyperplasia. This hyper-production of mucus may decrease nutrient absorption, underlying impaired growth in S. salmonis infected fish. I developed an in vitro plasma incubation test to predict host susceptibility of rainbow trout, carp, and sturgeon. The test showed the hierarchy of resistance of S. salmonis in sturgeon > carp > rainbow trout; this parallels epizootiological data. My research yielded new diagnostic tool, suggested new treatment target, improved in vitro conditions, and new model system for multi-functionality of flagella and flagellar signalling.