Free-living amoebae and other neglected protistan pathogens: Health emergency signals?

Protistan parasites have an undisputed global health impact. However, outside of a few key exceptions, e.g. the agent of malaria, most of these infectious agents are neglected as important health threats. The Symposium entitled “Free-living amoebae and neglected pathogenic protozoa: health emergency signals?” held at the European Congress of Protistology in Rome, July 2019, brought together researchers addressing scientific and clinical questions about some of these fascinating organisms. Topics presented included the molecular basis of pathogenicity in Acanthamoeba; genomics of Naegleria fowleri; and epidemiology of poorly diagnosed enteric protistan species, including Giardia, Cryptosporidium, Blastocystis, Dientamoeba. The Symposium aim was to excite the audience about the opportunities and challenges of research in these underexplored organisms and to underline the public health implications of currently under-appreciated protistan infections. The major take home message is that any knowledge that we gain about these organisms will allow us to better address them, in terms of monitoring and treatment, as sources of future health emergencies.     

A Simple Method For Plating and Cloning Ciliates and Other Protozoa

SYNOPSIS. A simple method is described for plating and cloning ciliates and other protozoa, based on a principle differing from that traditionally used for plating and cloning bacteria and other microorganisms. This procedure, referred to as the silicone-oil-plating-procedure (SOPP), involves vortexing small volumes of culture medium containing protozoa with larger volumes of a non-toxic silicone oil and plating the resulting unstable emulsion in small plastic petri plates. Discrete microdroplets of culture medium form containing protozoa entrapped and immobilized between the hydrophobic surfaces of the plastic petri dish and the oil. Protozoa, isolated by this method grow, divide, and multiply to form clones. the procedure may be used for plating and cloning protozoa in bacterized and axenic culture. Variations of the basic method may be applied to isolating protozoa from the wild, washing protozoa to remove microorganisms, screening for potential mutants, and for replica plating.     

Comparison of growth efficiencies of protozoa growing on bacteria deposited on surfaces and in suspension

Bacteria were deposited in tubes as compact pellets by centrifuging suspensions of cultured Vibrio at stationary phase. Numbers and protein biomass of flagellates added to these tubes and of the Vibrio, were followed and compared with the growth of the same and other protists on identical, uncentrifuged Vibrio. The flagellates Bodo saliens and Caecitellus parvulus, which could not be seen to multiply in tubes of suspended bacteria, grazed deposited bacteria actively as did the more versatile flagellate Cafeteria roenbergensis. The growth of these flagellates and their consumption of deposited bacteria were very similar to those of the flagellate Pteridomonas danica or the ciliate Uronema marinum fed with suspended bacteria, although deposit-feeders grew more slowly. Gross growth efficiencies (30-60%) of deposit-feeding flagellates were similar to those of the suspension-feeding protists. Caecitellus consumed 55 Vibrio to produce one flagellate, while 4,500 Vibrio were consumed to produce one Uronema. Surface-feeding flagellates are shown to be efficient bacterivores, capable of restricting the numbers of bacteria deposited on surfaces just as other protozoa control numbers of suspended bacteria.     

An Under-appreciated Component of Biodiversity in Plankton Communities: The Role of Protozoa in Lake Michigan (A Case Study)

Recent technological advances have led to the discovery that free-living, planktonic protozoa are ubiquitous in nature and appear to be important components of pelagic food webs (e.g., fluorescent straining, flow cytometry). Despite this, limited information exists tying their seasonality to rate processes that drive succession patterns. The abundance, and seasonal growth and grazing loss of an entire protozoan assemblage were evaluated in Lake Michigan. The protozoan assemblage was species-rich (100 taxa) and abundant throughout the year in Lake Michigan. Nano-sized protozoa (Hnano and Pnano, <20 μm in size) ranged in abundance from 10² to 10³ cells ml⁻¹, while micro-protozoa (Hmicro and Pmico, >20 and <200 μm in size) ranged in abundance from 4 to 17 cells ml⁻¹. The biomass of Hnano and Hmicro by itself represented more than 70–80% of crustacean zooplankton biomass, while Pnano and Pmicro constituted nearly 50% of phytoplankton biomass. Protozoa exhibited growth rates comparable to other components of the plankton in Lake Michigan, and some populations grew at rates similar to maximum rates determined in the laboratory (rates of 1–2 day⁻¹). Overall, it appears that macro-zooplankton predation is a major loss factor counter-balancing growth with only small differences between the two rate processes (<0.1 day⁻¹). Discrepancies between growth and grazing loss in the spring were likely attributed to sedimentation losses for larger species of tintinnids and dinoflagellates (Codonella, Tintinnidium, and Gymnodinium) that can account for their occurrence in the deep chlorophyll layer. In the summer, carnivory among similar sized species (Chromulina and small ciliates) may be additional loss factors impinging on the protozoan assemblage.     

Role of the protozoan Isotricha prostoma, liquid-, and solid-associated bacteria in rumen biohydrogenation of linoleic acid

From the simultaneous accumulation of hydrogenation intermediates and the disappearance of Isotricha prostoma after algae supplementation, we suggested a role of this ciliate and/or its associated bacteria in rumen biohydrogenation of unsaturated fatty acids. The experiments described here evaluated the role of I. prostoma and/or its associated endogenous and exogenous bacteria in rumen biohydrogenation of C18:2n-6 and its main intermediates CLA c9t11 and C18:1t11. Fractions of I. prostoma and associated bacteria, obtained by sedimentation of rumen fluid sampled from a monofaunated sheep, were used untreated, treated with antibiotics or sonicated to discriminate between the activity of I. prostoma and its associated bacteria, the protozoan or the bacteria, respectively. Incubations were performed in triplicate during 6 h with unesterified C18:2n-6, CLA c9t11 or C18:1t11 (400 μg/ml) and 0.1 g glucose/cellobiose (1/1, w/w). I. prostoma did not hydrogenate C18:2n-6 or its intermediates whereas bacteria associated with I. prostoma converted a limited amount of C18:2n-6 and CLA c9t11 to trans monoenes. C18:1t11 was not hydrogenated by either I. prostoma or its associated bacteria but was isomerized to C18:1c9. A phylogenetic analysis of clones originating from Butyrivibrio-specific PCR product was performed. This indicated that 71% of the clones from the endogenous and exogenous community clustered in close relationship with Lachnospira pectinoschiza. Additionally, the biohydrogenation activity of solid-associated bacteria (SAB) and liquid-associated bacteria (LAB) was examined and compared with the activity of the non-fractioned I. prostoma monofaunated rumen fluid (LAB + SAB). Both SAB and LAB were involved in rumen biohydrogenation of C18:2n-6. SAB fractions performed the full hydrogenation reaction to C18:0 while C18:1 fatty acids, predominantly C18:1t10 and C18:1t11, accumulated in the LAB fractions. SAB and LAB sequence analyses were mainly related to the genera Butyrivibrio and Pseudobutyrivibrio with 12% of the SAB clones closely related to the C18:0 producing B. proteoclasticus branch. In conclusion, this work suggests that I. prostoma and its associated bacteria play no role in C18:2n-6 biohydrogenation, while LAB convert C18:2n-6 to a wide range of C18:1 fatty acids and SAB produce C18:0, the end product of rumen lipid metabolism.     

Abundance and feeding of microheterotrophic flagellates from a eutrophic lake

The growth of planktonic bacteria from a eutrophic lake was evaluated with microflagellate predators present and absent. Differential filtration (50 and 8 µm filters) was used to exclude ciliates and larger zoo-and phytoplankton from replicate experimental cultures. Additional filtration (1 µm filter) excluded heterotrophic microflagellates from a second set of experimental cultures, producing cultures that contained either bacteria and microflagellates or only bacteria. Growth of bacteria and microflagellates was evaluated by epifluorescent microscopy from repeated sampling over approximately 200 h. Bacterial numbers were reduced in the presence of microflagellates, and microflagellates were observed to contain bacterial prey. However, microflagellate numbers were high (about 10⁶ cells ml^-1) and were less than an order of magnitude lower than bacterial numbers. Bacteria growing in the presence of microflagellates did not show predator-prey population oscillations but had in-phase oscillations in numbers, suggesting that microflagellate predation in freshwater may not control numbers of planktonic bacteria. Clearance rates of heterotrophic microflagellates, estimated to be only 30 body volumes hr^-1, were insufficient to maintain flagellate growth, suggesting that other energy sources may be needed to maintain microflagellates in eutrophic freshwater ecosystems.     

Hydrogenosomes in some anaerobic protozoa resemble mitochondria

Abstract Microbodies in six anaerobic ciliated protozoa ( Metopus, Brachonella, Plagiopyla, Parablepharisma, Sonderia, Saprodinium ) were found to be enclosed by two membranes. The inner membrane showed extensive infolding, division stages were observed, and in all genera apart from Sonderia and Parablepharisma , the microbodies contained an hydrogenase and were attached to methanogenic bacteria. Some of these ciliates are related to aerobic species with mitochondria. We believe that the microbodies are hydrogenosomes, that they are derived from mitochondria, and that their biochemical modification has incurred little change in the original mitochondrial ultrastructure. These observations weaken the case for the independent origin of hydrogenosomes from anaerobic prokaryotes.     

Evolution of the hydrogenosome

Since its discovery almost 25 years ago the enigmatic hydrogenosome, a redox organelle of anaerobic unicellular eukaryotes, has puzzled evolutionists as to its origin and function. Synthesis of recent molecular, physiological and morphological studies now favours the hypothesis that hydrogenosomes derived from a modification of pre-existing mitochondria, and argues against the previously held view that the hydrogenosome had a polyphyletic origin. These data provide evidence for a more ancient origin of mitochondria than hitherto thought.     

Early Evolution of Protists Introductory Remarks of Chairman

SYNOPSIS. Phylogenetic relations are ultimately determined by homologous relationships, including both structural and functional data. Following the establishment of those relationships, the direction of evolutionary change must be determined using paleontologic, developmental, and especially morphocline, data. From that perspective the direction of subsequent development becomes clearer and the problems of origins become more explicit. Using the foregoing methodology it has been possible to identify plausibly monophyletic groups of animal protists or protozoa. Allowing for attendant difficulties, there nevertheless emerges certain fairly convincing generalities: (a) the predominantly pseudopodial forms, with a few minor exceptions, have direct origins from apochlorotic algae; (b) the predominantly kinetidal forms (zooflagellates and ciliates), though also derived originally from apochlorotic algae, give evidence of extended evolutionary development with the especially noteworthy emergence of a permanent ingestatory structure; (c) both groups have increased size, a tendency towards multinuclearity and polyploidy, cytoplasmic differentiations of various sorts, and complex life cycles. In terms of further evolution, namely the emergence of multicellular animals, the pseudopodial forms are almost certainly a dead end while the kinetidal forms are arguably the ancestors of at least 2 metazoan phyla.     

Fractionation of the gene-size macronuclear chromatin fragments of the binucleated eukaryote Oxytricha

Summary The macronuclear chromatin of Oxytrichia nova consists of chromatin fragments which are fully soluble in 0.2 mM EDTA and whose DNA length varies from 500–25 000 bp. The DNA migrates electrophoretically as a series of discrete bands, with specific genes present in only one or a few bands. The chromatin fragments are composed of nucleosomes and migrate electrophoretically in proportion to their DNA length. These results suggest schemes for the fractionation of undigested chromatin in order to enrich for specific genes, facilitating analysis of changes in chromatin structure associated with changes in gene expression.     

In vitro antimalarial activity and molecular docking analysis of 4-aminoquinoline-clubbed 1,3,5-triazine derivatives

Aims: Present report describes the in vitro antimalarial activity and docking analysis of seven 4‐aminoquinoline‐clubbed 1,3,5‐triazine derivatives on pf‐DHFR‐TS. Methods and Results: The antimalarial activity was evaluated in vitro against chloroquine‐sensitive 3D7 strain of Plasmodium falciparum. Compounds were docked onto the active site of pf‐DHFR‐TS using docking server to explicate necessary structural requirements for antimalarial activity. Conclusion: Title molecules demonstrated considerable bioactivity against the malaria parasite. Docking analysis revealed deep engulfment of the molecules into the inner groove of pf‐DHFR‐TS active site by making stable ligand–receptor posses. Hydrophobic interaction was identified as the only major interacting force playing a role between ligand–receptor interaction and minor with hydrogen bonds. Signi?cance and Impact of the study: The study provided the novel insight into the necessary structural requirement for rationale‐based antimalarial drug discovery.     

Ultrastructural alterations in ciliated protozoa under heavy metal exposure

Transmission electron microscopy was used to study the ultrastructural changes induced by exposure to Cd or Zn in three species of ciliated protozoa: Colpoda steinii, Cyrtolophosis elongata and Drepanomonas revoluta. The main cytoplasmic alterations were partial mitochondrial degeneration, cytoplasmic vacuolisation, accumulation of membranous debris and autophagosome formation. At the nuclear level we detected nucleolar fusion in the macronucleus, and micronuclear membrane modifications. We compared these modifications with those coinciding with ciliate encystment (a differentiation process induced by environmental nutritional stress) and with changes in eukaryotic cells treated with staurosporine, a potent protein kinase inhibitor considered to be an apoptosis inducer. Exposure to heavy metals also coincided with the appearance of electron-dense accumulations in the cytoplasm, which might be related to metallothionein-mediated detoxification. The results are compared with previously reported data from ciliates and microalgae treated with heavy metals.     

Babesia argentina: the infectivity and immunogenicity of irradiated blood parasites for splenectomized calves

The intraerythrocytic forms of Babesia argentina were exposed to various doses of γ-radiation and then inoculated intravenously into splenectomized calves to study infectivity and immunogenicity of the irradiated parasites. Commencing with a dose of 8 krads, the proportion of organisms capable of multiplication in the host was progressively reduced until complete inhibition of multiplication was obtained with doses in the range of 50–70 krads. After exposures between 20 and 50 krads, the organisms showed reduced rates of multiplication in the host and produced only mild infections that left the surviving recipient animals strongly immune to reinfection. Parasites, completely inhibited by irradiation, did not protect splenectomized calves against challenge infection and behaved immunologically as killed organisms.     

中国典型地带土壤原生动物:Ⅱ生态学研究

研究了中国典型地带土壤原生动物生态学。结果表明,在各典型地带中,凋落物层原生动物的年平均现存量以温带（吉林长白山）和暖温带（北京小龙门）的为最大,北热带（云南西双版纳）的次之,中热带（海南尖峰岭）的最小;０～５ｃｍ土壤层的原生动物年平均现存量以暖温带和北热带的为最大,亚热带（武昌珞珈山）和温带的次之,高寒带（青海海北）和中热带的最小。各地带各样点凋落物层的原生动物现存量均大于０～５ｃｍ土壤层的。从丰度来看,在原生动物三大类群中,肉足虫现存量最大,鞭毛虫次之,纤毛虫现存量最小。各地带各样点的原生动物丰度高峰一般都出现在夏季。回归分析结果表明,土壤含水量、温度、ｐＨ值和有机质、总氮、总磷及总钾含量对土壤原生动物丰度均有显著影响。     

Environmental amoebae do not support the long‐term survival of virulent mycobacteria

Aims

To test the hypothesis that Mycobacterium bovis can persist in the environment within protozoa.

Methods and Results

In this study, we used a novel approach to detect internalized mycobacteria in environmental protozoa from badger latrines. Acid‐fast micro‐organisms were visualized in isolated amoebae, although we were unable to identify them to species level as no mycobacteria were grown from these samples nor was M. bovis detected by IS6110 PCR. Co‐incubation of Acanthamoeba castellanii with virulent M. bovis substantially reduced levels of bacilli, indicating that the amoebae have a negative effect on the persistence of M. bovis.

Conclusions

The internalization of mycobacteria in protozoa might be a rare event under environmental conditions. The results suggest that amoebae might contribute to the inactivation of M. bovis rather than representing a potential environmental reservoir.

Significance and Impact of the Study

Protozoa have been suggested to act as an environmental reservoir for M. bovis. The current study suggests that environmental amoebae play at most a minor role as potential reservoirs of M. bovis and that protozoa might inhibit persistence of M. bovis in the environment.     

The structural organization of the pathogenic protozoan Tritrichomonas foetus as seen in replicas of quick frozen,freeze-fractured and deep etched cells

Summary— The quick-freezing and freeze-etching technique was used to analyse the cytoskeleton of Tritrichomonas foetus, a pathogenic protozoan of the urogenital tract of cattle. The cytoplasm presented a network of filamentous structures interacting with each other, with the surface of the hydrogenosomes and the nuclear membrane. Two nm wide filamentous structures were found in the luminal space of the Golgi complex, connecting the two faces of each cisterna. The microtubules of the pelta-axostyle system were connected by bridges 30–40 nm long and 10 nm wide, regularly spaced with an interval of 25 nm. The costa is a structure formed by a complex array of filaments and globous structures. It seems to be connected to the recurrent flagellum through a complex network formed by 15 and 10 nm wide filaments which emerge from the peripheral region of the costa and penetrate into the surface projections of the protozoan body to which the recurrent flagellum is attached. Other filaments were seen connecting the surface of these projections with the surface of the flagellum.     

A New Heterolobosean Amoeboflagellate,Tetramitus dokdoensis n. sp., Isolated from a Freshwater Pond on Dokdo Island in the East Sea,Korea

The genus Tetramitus is a representative amoeboflagellate group within the Heterolobosea, and currently contains over a dozen species. Here, a new heterolobosean amoeboflagellate was isolated from a freshwater pond on Dokdo Island, Korea. The amoebae have eruptive pseudopodia, no uroidal filament, and a nucleus with a central nucleolus. The length and width of the amoebae are 15.5–28.0 μm and 5.4–12.6 μm, respectively. The flagellates are conical, with 4 flagella of equal length (~10 μm). There is a discrete rostrum in the subapical region of the flagellate form. The cyst has thin endo‐ and ectocyst layers and no cyst pores. The amoeba shows slow movement at 37 °C, but does not move at 42 °C under a light microscope. Phylogenies of the 18S rRNA gene and the ITS1‐5.8S rRNA gene‐ITS2 sequence show that the strain belongs to a subclade of Tetramitus that includes Tetramitus rostratus, Tetramitus waccamawensis and Tetramitus entericus, amongst others. Nonetheless, the strain is distinct from other species in both molecular phylogenetic trees. Thus the strain isolated from the Dokdo Island is proposed as a novel species, Tetramitus dokdoensis n. sp.     

Ciliate evolution: The ribosomal phylogenies of the tetrahymenine ciliates

Summary We have assembled and analyzed nucleotide sequences for several different rRNA components from tetrahymenine ciliates. These include previously published and some new 5S and 5.8S rRNAs for a total of 18 species. We also report sequences for some 30 species obtained by primer extension analysis of a region near the 5′ end of the 23S rRNAs (region 580). Phylogenetic trees have been constructed for these species, utilizing heuristics (shifting ditypic site analysis) described in a companion paper. The trees based on these sequences are consistent with each other and with those based on longer sequences of the 17S rRNA. They show the tetrahymenines to consist of a number of distinctive clusters of species. The clusters (ribosets) are homogeneous with respect to certain life history characteristics, especially the mode of mating type determination, but are inhomogeneous with respect to some morphological and life history features, such as cyst formation and adaptations to parasitism or carnivory. Using the same molecular data, we also begin to explore the relationships of the tetrahymenines to some other ciliate taxa and to some other protists.