Life cycle, biochemistry and chemotherapy of Spironucleus vortens

Spironucleus is an opportunistic protozoan parasite capable of causing devastating losses in the production of both ornamental and food fish. Control of infection outbreaks is problematic due to restrictions on the use of chemotherapeutics and rapid parasite transmission amongst fish. This PhD investigated the life cycle, biochemistry and chemotherapy of Spironucleus vortens. Direct transmission of S. vortens was found to be facilitated by the trophozoite form, information which may be applied in aquaculture to prevent infection outbreaks. No S. vortens cysts were observed in vitro or in vivo and trophozoites were able to survive for prolonged periods in the faeces of angelfish. This novel finding facilitated development of a non-invasive method to quantify the degree of intestinal colonization in the host, which was then applied to determine the efficacy of new and existing chemotherapeutics against S. vortens in vivo. Garlic-derived compounds were shown to be realistic alternatives to the current drug of choice, metronidazole, in the treatment of Spironucleosis in fish. Synergy between metronidazole and the garlic-derived compound, ajoene, was also observed in vitro and in vivo. The mode of action of metronidazole and garlic-derivatives involved disruption of S. vortens intracellular redox balance, a pivotal cellular process which ensures normal cellular function and survival. Further biochemical investigations into the antioxidant defence system (consisting of glutathione, thioredoxin and superoxide dismutase) as well as the carbohydrate and amino acid metabolism of S. vortens provided greater understanding of the success of this organism as a parasite. This is reflected in its ability to withstand fluctuations in O2 and nutrition during key pathogenic stages of its life cycle, including extra-intestinal systemic infection and transmission to a new host

Intracellular oxygen: similar results from two methods of measurement using Phosphorescent nanoparticles

The ability to resolve the spatio-temporal complexity of intracellular O2 distribution is the "Holy Grail" of cellular physiology. In an effort to obtain a minimally invasive approach to the mapping of intracellular O2 tensions, two methods of phosphorescent lifetime imaging microscopy were compared in the current study and gave similar results. These were two-photon confocal laser scanning microscopy with pinhole shifting, and picosecond time-resolved epi-phosphorescence microscopy using a single 0.5 μm focused spot. Both methods utilized Ru coordination complex embedded nanoparticles (45 nm diameter) as the phosphorescent probe, excited using pulsed outputs of a titanium–sapphire Tsunami lasers (710–1050 nm)

Water soluble, cyclometalated Pt(II)–Ln(III) conjugates towards novel bimodal imaging agents

Facile conjugation of a luminescent cyclometalated PtII complex with a DO3A-derived GdIII moiety yields a hybrid species with visible luminescence and enhanced relaxivity

Comparative biochemistry of Giardia, Hexamita and Spironucleus: Enigmatic diplomonads

The diplomonad genera are here represented by three highly diverse species, both free-living (Hexamita inflata), and parasitic (Spironucleus vortens and Giardia intestinalis). All three are moderately aerotolerant flagellates, inhabiting environments where O2 tensions are low and fluctuating. Many diplomonads are opportunistic pathogens of avian, terrestrial and aquatic animals. Hexamitids inhabit deep waters and sediments of lakes and marine basins, S. vortens commonly infects the intestinal tract of ornamental fish, particularly of cichlids and cyprinids, and G. intestinalis, the upper intestinal tracts of humans as well as domestic and farm animals. Despite these very different habitats, their known physiological and biochemical characteristics are similar, but they do differ in significant respects as their lifestyles and life cycles demand. They have efficient O2 scavenging systems, and are highly effective at countering rapid O2 fluctuations, or clustering away from its source (except for G. intestinalis when attached to the jejunal villi). Their core metabolic pathways (glycolysis using pyrophosphate), incomplete tricarboxylic acid cycle (lacking α-ketoglutarate dehydrogenase), and amino acid metabolism (with an alternative energy-generating arginine dihydrolase pathway as a possibility in some cases), largely conform to those of other protists inhabiting low-O2 environments. Mitochondrial evolutionary reduction to give hydrogenosomes as seen in Spironucleus spp. has proceeded further to its minimal state in the mitosomes of G. intestinalis. Understanding of essential redox reactions and the maintentence of redox state, especially in the infective encysted stage of G. intestinalis provide increasing possibilities for parasite control. To this aim a plethora of new synthetic chemicals and natural products (especially those from garlic, Allium sativum) show promise as replacements for the highly effective (but potentially toxic to higher organisms) 5-nitroimidazoles (e.g., metronidazole) in the treatment and/or prevention of dimplomonad infection in humans and animals

Unexpected properties of NADP-dependent secondary alcohol dehydrogenase (ADH-1) in Trichomonas vaginalis and other microaerophilic parasites

Our previous observation that NADP-dependent secondary alcohol dehydrogenase (ADH-1) is down-regulated in metronidazole-resistant Trichomonas vaginalis isolates prompted us to further characterise the enzyme. In addition to its canonical enzyme activity as a secondary alcohol dehydrogenase, a pronounced, so far unknown, background NADPH-oxidising activity in absence of any added substrate was observed when the recombinant enzyme or T. vaginalis extract were used. This activity was strongly enhanced at low oxygen concentrations. Unexpectedly, all functions of ADH-1 were efficiently inhibited by coenzyme A which is a cofactor of a number of key enzymes in T. vaginalis metabolism, i.e. pyruvate:ferredoxin oxidoreductase (PFOR). These observations could be extended to Entamoeba histolytica and Tritrichomonas foetus, both of which have a homologue of ADH-1, but not to Giardia lamblia which lacks an NADP-dependent secondary alcohol dehydrogenase. Although we could not identify the substrate of the observed background activity, we propose that ADH-1 functions as a major sink for NADPH in microaerophilic parasites at low oxygen tension

Antioxidant defences of Spironucleus vortens: Glutathione is the major non-protein thiol

The aerotolerant hydrogenosome-containing piscine diplomonad, Spironucleus vortens, is able to withstand high fluctuations in O2 tensions during its life cycle. In the current study, we further investigated the O2 scavenging and antioxidant defence mechanisms which facilitate the survival of S. vortens under such oxidizing conditions. Closed O2 electrode measurements revealed that the S. vortens ATCC 50386 strain was more O2 tolerant than a freshly isolated S. vortens intestinal strain (Sv1). In contrast to the related human diplomonad, Giardia intestinalis, RP-HPLC revealed the major non-protein thiols of S. vortens to be glutathione (GSH, 776 nmol/107 cells) with cysteine and H2S as minor peaks. Furthermore, antioxidant proteins of S. vortens were assayed enzymatically and revealed that S. vortens possesses superoxide dismutase and NADH oxidase (883 and 37.5 nmol/min/mg protein, respectively), but like G. intestinalis, lacks catalase and peroxidase activities. Autofluorescence of NAD(P)H and FAD alongside the fluorescence of the GSH-adduct in monochlorobimane-treated live organisms allowed the monitoring of redox balances before and after treatment with inhibitors, metronidazole and auranofin. H2O2 was emitted into the exterior of S. vortens at a rate of 2.85 pmol/min/106 cells. Metronidazole and auranofin led to depletion of S. vortens intracellular NAD(P)H pools and an increase in H2O2 release with concomitant oxidation of GSH, respectively. Garlic-derived compounds completely inhibited O2 consumption by S. vortens (ajoene oil), or significantly depleted the intracellular GSH pool of the organism (allyl alcohol and DADS). Hence, antioxidant defence mechanisms of S. vortens may provide novel targets for parasite chemotherapy

Non-invasive investigation of Spironucleus vortens transmission in freshwater angelfish Pterophyllum scalare

Spironucleus vortens is a protozoan fish parasite of veterinary and economic importance in the ornamental aquaculture industry. Despite this, key aspects of the life cycle of this organism, including its mode of transmission, have not been fully elucidated. We developed a non-invasive method for quantifying S. vortens in freshwater angelfish, which was then used to investigate parasite transmission and aggregation within host populations. As previously observed for S. meleagridis and S. salmonis, motile S. vortens trophozoites were detected in host faeces using light microscopy. Species-level identification of these flagellates was confirmed using 16S rDNA PCR. Faecal trophozoite counts were significantly correlated with trophozoite counts from the posterior intestine, the primary habitat of the parasite. This novel finding allowed effective prediction of intestinal parasite load from faecal counts. Overall, faecal count data revealed that 20% of hosts harbour 83% of parasites, conforming to the Pareto Principle (80/20 rule) of parasite aggregation with implications for parasite transmission. Trophozoites survived for ≥36 d outside the host within faeces and remained motile at low pH (comparable with that of angelfish stomach). No putative S. vortens cysts were observed in cultures or faecal samples. This calls into question the commonly accepted hypothesis that a protective cyst is required in the life cycle of S. vortens to facilitate transmission to a new host