Cryptobia iubilans Infections in Discus Fish in Trinidad and Tobago.

Discus (Symphysodon spp.) are costly and prized specimens in the international ornamental fish trade. The majority of discus submitted to the Aquatic Animal Health Unit at the University of the West Indies School of Veterinary Medicine for necropsy between September 2010 and September 2015 had lesions consistent with Cryptobia iubilans infection, thus prompting this study. To determine the prevalence of the flagellated gastrointestinal protozoan C. iubilans in discus fish, 32 discus were sourced from 10 suppliers, including breeders, importers, and hobbyists across Trinidad. Fish were euthanized, and the internal organs, particularly the stomach and intestine, were observed under a light microscope for characteristic granulomatous lesions and/or live C. iubilans parasites. All wet-mount slides on which granulomas were observed were also Ziehl-Neelsen acid-fast stained to presumptively exclude the presence of Mycobacterium spp., the main differential when diagnosing C. iubilans-associated granulomatous gastritis or to determine the presence of dual infections. Further histological analyses were performed on stomach and intestinal sections, and transmission electron microscopy was used to confirm the parasite in stomach sections. The prevalence of C. iubilans infection was found to be 81.3%, and the prevalence of presumptive dual infections with Mycobacterium spp. was found to be 21.9%. To the best of our knowledge, this is the first documented study of C. iubilans infections in the wider Caribbean region.

Paramoeba aparasomata n. sp., a symbiont-free species, and its relative Paramoeba karteshi n. sp. (Amoebozoa, Dactylopodida).

Two brackish water amoebae have been isolated and studied from the benthic biotopes of the Chupa Inlet (Kandalaksha Bay, northwestern Russia). Both strains can be identified as new species of the genus Paramoeba (Amoebozoa, Dactylopodida, Paramoebidae) based on light microscopical characters, structure of microscales on the cell surface and molecular evidence based on the analyses of two genes, nuclear SSU rRNA and mitochondrial cytochrome c oxidase subunit 1 (COI). Paramoeba aparasomata n. sp. is of particular interest because this amoeba is permanently lacking a symbiotic Perkinsela-like organism (PLO) present in other species of Paramoeba and Neoparamoeba. The results obtained show that scaly dactylopodial amoebae lacking PLO are not necessarily members of Korotnevella. In particular, we suggest that Korotnevella nivo Smirnov, 1997, with microscales very similar to those of Paramoeba eilhardi and the species studied here in structure, may be in fact a member of Paramoeba. Molecular data on K. nivo have to be obtained and analysed to test this hypothesis. Based on our new results we emend the diagnosis of the genus Paramoeba to make it more fit to the current phylogenetic conception.

Sensing What's Out There - Kinetoplastid Parasites.

Kinetoplastid parasites such as trypanosomes and Leishmania must adapt to their environments to survive within their hosts, yet they do not express many of the well established families of signal transduction receptors. Evidence suggests that other membrane proteins, including transporters and channels, play central roles in environmental sensing in these parasites.

Gene Function Discovery for Kinetoplastid Pathogens.

We propose to integrate the existing and new experimental data with computational tools to model interaction networks for the most prominent kinetoplastid pathogens. These interaction networks will vastly expand the functional annotation of the kinetoplastid genomes, which in turn are critical for identifying new routes of disease intervention.

Measurement of Tunic Hardness in an Edible Ascidian, Halocynthia roretzi, with Remarks on Soft Tunic Syndrome.

The infection caused by a kinetoplastid flagellate, Azumiobodo hoyamushi, in an ascidian, Halocynthia roretzi, results in softening of the tunic, and finally death. This disease is usually recognized using palpation of the softening tunic, and A. hoyamushi infection is detectable using microscopy or PCR amplification of specific gene fragments. The present study is the first quantitative evaluation of the symptoms of soft tunic syndrome by measuring the amount of bending (bending) and the peak force required to pierce the tunic (force). There was a strong correlation between bending and force. Correlation analyses among other parameters (ascidian total weight, tunic thickness, and tunic water content) indicated that larger ascidians had harder and thicker tunics with a higher water content. As compared to the tunic of healthy individuals, softened tunic was thinner and had lower water content. Infected tunics thus possibly lose water and become softer and thinner. Mechanisms for maintaining the appropriate water level content may be crucial for preventing tunic softening.

Tunic extract of the host ascidian attracts the causal agent of soft tunic syndrome, Azumiobodo hoyamushi (Kinetoplastea: Neobodonida).

Azumiobodo hoyamushi, a kinetoplastid flagellate, is the causative agent of soft tunic syndrome, an infectious disease of the edible ascidian Halocynthia roretzi. The flagellate is thought to invade the tunic matrix via a damaged area of the tunic on the siphon wall. We hypothesized that the flagellate locates the tunic entry site by a chemotactic response to soluble substances diffused from the host ascidians. To investigate this hypothesis, we examined whether the flagellate shows a chemotactic response to tissue extracts (tunic and other tissues) from the host ascidian H. roretzi. We tested extracts from 5 tissues as well as hemolymph. Only the tunic extract showed significant positive chemotactic activity, and the activity decreased with increasing dilution. Furthermore, autoclaved tunic extract, extracts from diseased individuals, and extract from the styelid ascidian Styela clava also had chemotactic activity, although the activities were lower than that of tunic extract from healthy H. roretzi. Ultrafiltration of the tunic extract through a 3 kDa cutoff membrane completely abrogated the activity; the ultrafiltration retentate still showed activity. Thus, the soluble factors that attract the flagellate are present exclusively in the tunic extract, and the chemotactic factors are larger than 3 kDa. Our experiments also suggested that the tunic extract contains both heat-stable and heat-labile factors. We conclude that the flagellate locates the tunic entry site by chemotaxis toward soluble factors that diffuse from a damaged area of the tunic on the siphon wall.

Feeding and growth of the marine heterotrophic nanoflagellates, Procryptobia sorokini and Paraphysomonas imperforata on a bacterium, Pseudoalteromonas sp. with an inducible defence against grazing.

Heterotrophic marine nanoflagellates are important grazers on bacteria in the water column. Some marine bacteria appear more resistant to grazing than do others. Marine nanoflagellates can be grown in the laboratory in batch cultures fed specific bacterial isolates. In some cultures, the flagellates appear unable to completely deplete the bacterial prey even when the bacterial strain otherwise is an excellent prey. This may indicate that some marine bacteria are able to induce defence mechanisms if they are grazed by nanoflagellates. Four morphologically distinct marine heterotrophic nanoflagellates, of which 3 were still identified as Procryptobia sorokini (Kinetoplastea) and one as Paraphysomonas imperforata (Chrysophyceae) were isolated from a coastal location along with 3 isolates of the marine bacterium Pseudoalteromonas sp. Flagellate growth and grazing on bacterial prey were analysed in batch cultures. Pseudoalteromonas was a suitable prey for all 4 flagellate isolates. They grazed and grew on Pseudoalteromonas as sole prey with maximal cell-specific growth rates of 0.1-0.25 h-1 and gross growth efficiencies of 38-61%. Exposure to dense flagellate cultures or their supernatants did, however, cause a fraction of the Pseudoalteromonas cells to aggregate and the bacterium became apparently resistant to grazing. Concentrations of suspended Pseudoalteromonas cells were therefore not decreased below 1,700-7,500 cells µL-1 by any of the flagellate isolates. These results indicate that Pseudoalteromonas sp. can be an excellent prey to marine nanoflagellates but also that is in possession of inducible mechanisms that protect against flagellate grazing.

Novel scaffolds for inhibition of Cruzipain identified from high-throughput screening of anti-kinetoplastid chemical boxes.

American Trypanosomiasis or Chagas disease is a prevalent, neglected and serious debilitating illness caused by the kinetoplastid protozoan parasite Trypanosoma cruzi. The current chemotherapy is limited only to nifurtimox and benznidazole, two drugs that have poor efficacy in the chronic phase and are rather toxic. In this scenario, more efficacious and safer drugs, preferentially acting through a different mechanism of action and directed against novel targets, are particularly welcome. Cruzipain, the main papain-like cysteine peptidase of T. cruzi, is an important virulence factor and a chemotherapeutic target with excellent pre-clinical validation evidence. Here, we present the identification of new Cruzipain inhibitory scaffolds within the GlaxoSmithKline HAT (Human African Trypanosomiasis) and Chagas chemical boxes, two collections grouping 404 non-cytotoxic compounds with high antiparasitic potency, drug-likeness, structural diversity and scientific novelty. We have adapted a continuous enzymatic assay to a medium-throughput format and carried out a primary screening of both collections, followed by construction and analysis of dose-response curves of the most promising hits. Using the identified compounds as a starting point a substructure directed search against CHEMBL Database revealed plausible common scaffolds while docking experiments predicted binding poses and specific interactions between Cruzipain and the novel inhibitors.

Taming Parasites by Tailoring Them.

The next-generation gene editing based on CRISPR (clustered regularly interspaced short palindromic repeats) has been successfully implemented in a wide range of organisms including some protozoan parasites. However, application of such a versatile game-changing technology in molecular parasitology remains fairly underexplored. Here, we briefly introduce state-of-the-art in human and mouse research and usher new directions to drive the parasitology research in the years to come. In precise, we outline contemporary ways to embolden existing apicomplexan and kinetoplastid parasite models by commissioning front-line gene-tailoring methods, and illustrate how we can break the enduring gridlock of gene manipulation in non-model parasitic protists to tackle intriguing questions that remain long unresolved otherwise. We show how a judicious solicitation of the CRISPR technology can eventually balance out the two facets of pathogen-host interplay.

Toward establishing model organisms for marine protists: Successful transfection protocols for Parabodo caudatus (Kinetoplastida: Excavata).

We developed protocols for, and demonstrated successful transfection of, the free-living kinetoplastid flagellate Parabodo caudatus with three plasmids carrying a fluorescence reporter gene (pEF-GFP with the EF1 alpha promoter, pUB-GFP with Ubiquitin C promoter, and pEYFP-Mitotrap with CMV promoter). We evaluated three electroporation approaches: (1) a square-wave electroporator designed for eukaryotes, (2) a novel microfluidic transfection system employing hydrodynamically-controlled electric field waveforms, and (3) a traditional exponential decay electroporator. We found the microfluidic device provides a simple and efficient platform to quickly test a wide range of electric field parameters to find the optimal set of conditions for electroporation of target species. It also allows for processing large sample volumes (>10 ml) within minutes, increasing throughput 100 times over cuvettes. Fluorescence signal from the reporter gene was detected a few hours after transfection and persisted for 3 days in cells transfected by pEF-GFP and pUB-GFP plasmids and for at least 5 days post-transfection for cells transfected with pEYFP-Mitotrap. Expression of the reporter genes (GFP and YFP) was also confirmed using reverse transcription-PCR (RT-PCR). This work opens the door for further efforts with this taxon and close relatives toward establishing model systems for genome editing.

Isolation of Novel Trypanosomatid, Zelonia australiensis sp. nov. (Kinetoplastida: Trypanosomatidae) Provides Support for a Gondwanan Origin of Dixenous Parasitism in the Leishmaniinae.

The genus Leishmania includes approximately 53 species, 20 of which cause human leishmaniais; a significant albeit neglected tropical disease. Leishmaniasis has afflicted humans for millennia, but how ancient is Leishmania and where did it arise? These questions have been hotly debated for decades and several theories have been proposed. One theory suggests Leishmania originated in the Palearctic, and dispersed to the New World via the Bering land bridge. Others propose that Leishmania evolved in the Neotropics. The Multiple Origins theory suggests that separation of certain Old World and New World species occurred due to the opening of the Atlantic Ocean. Some suggest that the ancestor of the dixenous genera Leishmania, Endotrypanum and Porcisia evolved on Gondwana between 90 and 140 million years ago. In the present study a detailed molecular and morphological characterisation was performed on a novel Australian trypanosomatid following its isolation in Australia's tropics from the native black fly, Simulium (Morops) dycei Colbo, 1976. Phylogenetic analyses were conducted and confirmed this parasite as a sibling to Zelonia costaricensis, a close relative of Leishmania previously isolated from a reduviid bug in Costa Rica. Consequently, this parasite was assigned the name Zelonia australiensis sp. nov. Assuming Z. costaricensis and Z. australiensis diverged when Australia and South America became completely separated, their divergence occurred between 36 and 41 million years ago at least. Using this vicariance event as a calibration point for a phylogenetic time tree, the common ancestor of the dixenous genera Leishmania, Endotrypanum and Porcisia appeared in Gondwana approximately 91 million years ago. Ultimately, this study contributes to our understanding of trypanosomatid diversity, and of Leishmania origins by providing support for a Gondwanan origin of dixenous parasitism in the Leishmaniinae.

Heme pathway evolution in kinetoplastid protists.

BACKGROUND: Kinetoplastea is a diverse protist lineage composed of several of the most successful parasites on Earth, organisms whose metabolisms have coevolved with those of the organisms they infect. Parasitic kinetoplastids have emerged from free-living, non-pathogenic ancestors on multiple occasions during the evolutionary history of the group. Interestingly, in both parasitic and free-living kinetoplastids, the heme pathway-a core metabolic pathway in a wide range of organisms-is incomplete or entirely absent. Indeed, Kinetoplastea investigated thus far seem to bypass the need for heme biosynthesis by acquiring heme or intermediate metabolites directly from their environment. RESULTS: Here we report the existence of a near-complete heme biosynthetic pathway in Perkinsela spp., kinetoplastids that live as obligate endosymbionts inside amoebozoans belonging to the genus Paramoeba/Neoparamoeba. We also use phylogenetic analysis to infer the evolution of the heme pathway in Kinetoplastea. CONCLUSION: We show that Perkinsela spp. is a deep-branching kinetoplastid lineage, and that lateral gene transfer has played a role in the evolution of heme biosynthesis in Perkinsela spp. and other Kinetoplastea. We also discuss the significance of the presence of seven of eight heme pathway genes in the Perkinsela genome as it relates to its endosymbiotic relationship with Paramoeba.

Disinfection of fertilized eggs of the edible ascidian Halocynthia roretzi for prevention of soft tunic syndrome.

Azumiobodo hoyamushi, the causative agent of soft tunic syndrome, was likely introduced to farming sites of the edible ascidian Halocynthia roretzi via ascidian spat. The source of infection is thought to be cysts of A. hoyamushi that reside in the substrates on which the ascidian spat are attached, but not the spat themselves. Thus, there is a need to develop methods to prevent contamination of the substrates with A. hoyamushi during seed production of the ascidian. We evaluated the protozoacidal effects of sodium hypochlorite and povidone-iodine against the flagellate and temporary cyst forms of A. hoyamushi. Additionally, we evaluated the effects of these disinfectants on the development of fertilized ascidian eggs. The flagellate form of A. hoyamushi was completely inactivated by povidone-iodine (5 ppm, 1 min) and sodium hypochlorite (1 ppm, 1 min). The temporary cysts of A. hoyamushi were completely inactivated by both disinfectants (5 ppm, 1 min). Disinfection with 50 ppm povidone-iodine for 15 min or 5 ppm sodium hypochlorite for 15 min had no effect on ascidian embryogenesis. Thus, horizontal transmission of A. hoyamushi via the substrates can be efficiently prevented by disinfecting ascidian eggs or tools used for spawning with povidone-iodine baths ranging from 5 ppm for 1 min to 50 ppm for 15 min without any side effects.

Flagellar membrane proteins in kinetoplastid parasites.

All kinetoplastid parasites, including protozoa such as Leishmania species, Trypanosoma brucei, and Trypanosoma cruzi that cause devastating diseases in humans and animals, are flagellated throughout their life cycles. Although flagella were originally thought of primarily as motility organelles, flagellar functions in other critical processes, especially in sensing and signal transduction, have become more fully appreciated in the recent past. The flagellar membrane is a highly specialized subdomain of the surface membrane, and flagellar membrane proteins are likely to be critical components for all the biologically important roles of flagella. In this review, we summarize recent discoveries relevant to flagellar membrane proteins in these parasites, including the identification of such proteins, investigation of their biological functions, and mechanisms of selective trafficking to the flagellar membrane. Prospects for future investigations and current unsolved problems are highlighted.

Flagellar motility in eukaryotic human parasites.

A huge variety of protists rely on one or more motile flagella to either move themselves or move fluids and substances around them. Many of these flagellates have evolved a symbiotic or parasitic lifestyle. Several of the parasites have adapted to human hosts, and include agents of prevalent and serious diseases. These unicellular parasites have become specialised in colonising a wide range of biological niches within humans. They usually have diverse transmission cycles, and frequently manifest a variety of distinct morphological stages. The motility of the single or multiple flagella plays important but understudied roles in parasite transmission, host invasion, dispersal, survival, proliferation and pathology. In this review we provide an overview of the important human pathogens that possess a motile flagellum for at least part of their life cycle. We highlight recently published studies that aim to elucidate motility mechanisms, and their relevance for human disease. We then bring the physics of swimming at the microscale into context, emphasising the importance of interdisciplinary approaches for a full understanding of flagellate motility - especially in light of the parasites' microenvironments and population dynamics. Finally, we summarise some important technological aspects, describing challenges for the field and possibilities for motility analyses in the future.

Cellulose is not degraded in the tunic of the edible ascidian Halocynthia roretzi contracting soft tunic syndrome.

Soft tunic syndrome is a fatal disease in the edible ascidian Halocynthia roretzi, causing serious damage to ascidian aquaculture in Korea and Japan. In diseased individuals, the tunic, an integumentary extracellular matrix of ascidians, softens and eventually tears. This is an infectious disease caused by the kinetoplastid flagellate Azumiobodo hoyamushi. However, the mechanism of tunic softening remains unknown. Because cellulose fibrils are the main component of the tunic, we compared the contents and structures of cellulose in healthy and diseased tunics by means of biochemical quantification and X-ray diffractometry. Unexpectedly, the cellulose contents and structures of cellulose microfibrils were almost the same regardless of the presence or absence of the disease. Therefore, it is unlikely that thinning of the microfibrils occurred in the softened tunic, because digestion should have resulted in decreases in crystallinity index and crystallite size. Moreover, cellulase was not detected in pure cultures of A. hoyamushi in biochemical and expressed sequence tag analyses. These results indicate that cellulose degradation does not occur in the softened tunic.

Encystment and excystment of kinetoplastid Azumiobodo hoyamushi, causal agent of soft tunic syndrome in ascidian aquaculture.

Soft tunic syndrome in the edible ascidian Halocynthia roretzi is caused by the kinetoplastid flagellate Azumiobodo hoyamushi, which was found to assume a fusiform cell form with 2 flagella in axenic, pure culture. When the flagellate form was incubated in sterilized artificial seawater (pH 8.4), some of the cells became cyst-like and adhered to the bottom of the culture plate. The cyst-like forms were spherical or cuboidal, and each had 2 flagella encapsulated in its cytoplasm. Encystment was also induced in culture medium alkalified to the pH of seawater (8.4) but not in unmodified (pH 7.2) or acidified media (pH 6.4). More than 95% of the cyst-like cells converted to the flagellate form within 1 d following transfer to seawater containing ascidian tunic extracts from host ascidians. The cyst-like cells were able to survive in seawater with no added nutrients for up to 2 wk at 20°C and for a few months at 5 to 15°C. The survival period in seawater depended on temperature: some cyst-like cells survived 3 mo at 10°C, and ca. 95% of these converted to flagellate forms in seawater containing tunic extracts. Thus, A. hoyamushi is able to persist under adverse conditions in a cyst-like form able to adhere to organic and inorganic substrata for protracted periods of time.

Quorum sensing-regulated chitin metabolism provides grazing resistance to Vibrio cholerae biofilms.

Association of Vibrio cholerae with chitinous surfaces of zooplankton is important for its persistence in marine environments, as it provides accessibility to nutrients and resistance to stresses. Predation by heterotrophic protists has a major impact on the survival of V. cholerae. V. cholerae forms biofilms as its main defensive strategy, and quorum sensing (QS) additionally regulates the production of antiprotozoal factors. The role of chitin and QS regulation in V. cholerae grazing resistance was investigated by exposing V. cholerae wild-type (WT) and QS mutant biofilms grown on chitin flakes to the bacteriotrophic, surface-feeding flagellate Rhynchomonas nasuta. V. cholerae formed more biofilm biomass on chitin flakes compared with nonchitinous surfaces. The growth of R. nasuta was inhibited by WT biofilms grown on chitin flakes, whereas the inhibition was attenuated in QS mutant biofilms. The chitin-dependent toxicity was also observed when the V. cholerae biofilms were developed under continuous flow or grown on a natural chitin source, the exoskeleton of Artemia. In addition, the antiprotozoal activity and ammonium concentration of V. cholerae biofilm supernatants were quantified. The ammonium levels (3.5 mM) detected in the supernatants of V. cholerae WT biofilms grown on chitin flakes were estimated to reduce the number of R. nasuta by >80% in add-back experiments, and the supernatant of QS mutant biofilms was less toxic owing to a decrease in ammonium production. Transcriptomic analysis revealed that the majority of genes involved in chitin metabolism and chemotaxis were significantly downregulated in QS mutant biofilms when grown on chitin compared with the WT biofilms.

Host specificity, pathogenicity, and mixed infections of trypanoplasms from freshwater fishes.

This work summarizes the results of the 8-year study focused on Trypanoplasma sp. parasitizing freshwater fishes in the vicinity of Kyiv, Ukraine. Out of 570 fish specimens of 2 different species analyzed, 440 individuals were found to be infected. The prevalence of infection ranged from 24 % in Abramis brama Linnaeus (freshwater bream) to 100 % in Cobitis taenia Linnaeus (spined loach). The level of parasitemia also varied between moderate in freshwater bream and very high in spined loach. Interestingly, no clinical manifestations of trypanoplasmosis were observed even in extremely heavily infected C. taenia. We hypothesize that different species may differ in evolutionary timing allowing for reciprocal adaptation of the members of the "host-parasite" system. Molecular analysis of the 18S rRNA sequences revealed that several specimens were simultaneously infected with at least two different trypanoplasm species. To the best of our knowledge, this is the first report of the mixed infection with fish trypanoplasms.

Quantitative assessment of Azumiobodo hoyamushi distribution in the tunic of soft tunic syndrome-affected ascidian Halocynthia roretzi using real-time polymerase chain reaction.

BACKGROUND: The kinetoplastid parasite, Azumiobodo hoyamushi, is the causative agent of soft tunic syndrome (STS) in ascidians and leads to their mass mortality in Korean waters. This study was conducted to quantify A. hoyamushi density during the development of STS in the tunics of ascidians (Halocynthia roretzi) using real-time polymerase chain reaction (qPCR). FINDINGS: The infection intensity of A. hoyamushi, as measured by qPCR, varied depending on the part of the tunic analyzed, as well as the stage of STS development. The highest infection intensity was recorded in the tunics of the siphons. The infection intensity of A. hoyamushi in the siphons was only 2.9 cell/tunic (area, 0.25 cm(2)) or 106.0 cell/gram tunic (GT) in the early phase of STS, but this value increased dramatically to 16,066 cells/tunic (0.25 cm(2)) or 617,004 cell/GT at the time of death. The number of A. hoyamushi parasites increased gradually and their distribution spread from the siphons to the other parts of the tunics. CONCLUSIONS: qPCR enabled the quantitation of A. hoyamushi and the results revealed that parasite density increased as STS progressed. In addition, our results suggested that the siphons might function as the portal of entry for A. hoyamushi during infection.