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International audience; AbstractCryptosporidium parvum is an enteric parasite and a major contributor to acute enteritis in calves worldwide, causing an important economic burden for farmers. This parasite poses a major public health threat through transmission between livestock and humans. Our previous pilot study in Western Europe revealed a high prevalence of Cryptosporidium in calves of dairy farms. In the sequel study herein, 936 faecal samples were collected from the same 51 dairy farms across Belgium, France, and the Netherlands. Following DNA extraction, Cryptosporidium screening was carried out using nested-PCR amplification targeting the SSU rRNA gene. All positive samples were sequenced, and phylogenetic analyses were used to identify the Cryptosporidium spp. present. The 60 kDa glycoprotein (gp60) gene was also sequenced to determine the C. parvum subtypes present. Prevalence of Cryptosporidium ranged from 23.3% to 25%, across the three countries surveyed. The parasite was found in most of the farms sampled, with 90.2% testing positive. Cryptosporidium parvum, C. bovis, C. ryanae and C. andersoni were all identified, with the former being the most predominant, representing 71.4% of all infections. Cryptosporidium parvum was associated with pre-weaned calves, while other species were associated with older animals. Subtyping of gp60 gene revealed nine subtypes, eight of which have previously been reported to cause clinical disease in humans. Similarly to the first study, vertical transmission was not a major contributor to Cryptosporidium spread. Our study highlights the need for further investigation into cryptosporidiosis transmission, and future studies will require a One Health approach to reduce the impact of this disease.

The present study aimed to estimate the prevalence and molecular characterization of Cryptosporidium spp. in six different fish species both from marine and freshwater environments.During a period of 2 years (2018-2020), a total of 415 fecal samples and 565 intestinal scrapings were collected in seven provinces from the central and eastern Algeria. From those, 860 fish belonged to six different species, two of which are cultured marine and four are wild freshwater fish. All samples were screened for Cryptosporidium spp. presence using molecular techniques. Nested PCR approach was performed to amplify partial sequences of the small subunit ribosomal RNA (SSU rRNA) and 60-kDa glycoprotein (GP60) genes for Cryptosporidium genotyping and subtyping. Detailed statistical analysis was performed to assess the prevalence variation of Cryptosporidium infection according to different risk factors.Nested PCR analysis of SSU gene revealed 173 Cryptosporidium positive fish, giving an overall prevalence of 20.11% (17.5-23.0). Cryptosporidium spp. was detected in 8.93% (42/470) of cultured marine fish and 33.58% (131/390) of wild freshwater fish. Overall, the prevalence was affected by all studied risk factors, except the gender. Molecular characterization and subtyping of Cryptosporidium isolates showed occurrence of IIaA16G2R1 and IIaA17G2R1 subtypes of C. parvum in the fish species Sparus aurata.The present study provides the first epidemiological data on the prevalence and associated risk factors of Cryptosporidium spp. in farmed marine and wild freshwater fish and the first molecular data on the occurrence of zoonotic C. parvum in fish from North Africa (Algeria).

Blastocystis is a protist of controversial pathogenicity inhabiting the gut of humans and other animals. Despite a century of intense study, understanding of the epidemiology of Blastocystis remains fragmentary. Here, we aimed to explore its prevalence, stability of colonisation and association with various factors in a rural elementary school in northern Thailand. One hundred and forty faecal samples were collected from 104 children at two time points (tp) 105 days apart. For tp2, samples were also obtained from 15 animals residing on campus and seven water locations. Prevalence in children was 67% at tp1 and 89% at tp2, 63% in chickens, 86% in pigs, and 57% in water. Ten STs were identified, two of which were shared between humans and animals, one between animals and water, and three between humans and water. Eighteen children (out of 36) carried the same ST over both time points, indicating stable colonisation. Presence of Blastocystis (or ST) was not associated with body mass index, ethnicity, birth delivery mode, or milk source as an infant. This study advances understanding of Blastocystis prevalence in an understudied age group, the role of the environment in transmission, and the ability of specific STs to stably colonise children.

Blastocystisis a stramenopile protist of controversial pathogenicity. The organism colonizes a broad range of vertebrate and invertebrate hosts.Blastocystishas also been found in the environment both in water and soil. Several routes of transmission have been proposed including human-to-human, animal-to-human and via contaminated food and water. In recent years, the presence ofBlastocystisin vegetables has started to be explored. However, most studies have focused on microscopic detection. Moreover, works of this type from Asia are barely available. Hence, the aim of this preliminary study was to examine the occurrence ofBlastocystisin raw vegetables sold in markets in northern Thailand. Fresh produce (n=20) commonly used in Thai cuisine (Lanna) was purchased from two street markets and screened forBlastocystisusing qPCR.Blastocystiswas detected in 45% of the samples with the dominant subtype being ST3. Produce growing underground, such as galangal, carrot and beetroot were positive for the organism suggesting soil or inadequately composted manure as the source of contamination. To our knowledge, our study is the first to perform subtyping ofBlastocystisin vegetables. Our results hint towards fresh produce being an, as yet, not widely explored, transmission route ofBlastocystisin the studied community. Looking forward, large-scale investigations should on the prevalence of this and other organisms under the One Health umbrella should be undertaken.

Blastocystis is a ubiquitous, widely distributed protist inhabiting the gastrointestinal tract of humans and other animals. The organism is genetically diverse, and so far, at least 28 subtypes (STs) have been identified with ST1–ST9 being the most common in humans. The pathogenicity of Blastocystis is controversial. Several routes of transmission have been proposed including faecal-oral (e.g. zoonotic, anthroponotic) and waterborne. Research on the latter has gained traction in the last few years with the organism having been identified in various bodies of water, tap water and rainwater collection containers including water that has been previously filtered and/or chlorinated. Herein, we assessed the resistance of 11 strains maintained in culture, spanning ST1–ST9 to various chlorine and hydrogen peroxide concentrations for 24 hours and performed recovery assays along with re-exposure. Following treatment with both compounds, all subtypes showed increased resistance, and viability could be visualised at the cellular level. These results are hinting at the presence of mechanism of resistance to both chlorine and hydrogen peroxide.

Blastocystis is a ubiquitous, widely distributed protist inhabiting the gastrointestinal tract of humans and other animals. The organism is genetically diverse, and so far, at least 28 subtypes (STs) have been identified with ST1–ST9 being the most common in humans. The pathogenicity of Blastocystis is controversial. Several routes of transmission have been proposed including fecal–oral (e.g., zoonotic, anthroponotic) and waterborne. Research on the latter has gained traction in the last few years with the organism having been identified in various bodies of water, tap water, and rainwater collection containers including water that has been previously filtered and/or chlorinated. Herein, we assessed the resistance of 11 strains maintained in culture, spanning ST1–ST9 to various chlorine and hydrogen peroxide concentrations for 24 h, and performed recovery assays along with re-exposure. Following the treatment with both compounds, all subtypes showed increased resistance, and viability could be visualized at the cellular level. These results are hinting at the presence of mechanism of resistance to both chlorine and hydrogen peroxide. As such, this pilot study can be the platform for developing guidelines for water treatment processes.

Chagas disease (CD) is a parasitic, systemic, chronic, and often fatal illness caused by infection with the protozoan

International audience; The alternative oxidase (AOX) is a protein involved in supporting enzymatic reactions of the Krebs cycle in instances when the canonical (cytochrome-mediated) respiratory chain has been inhibited, while allowing for the maintenance of cell growth and necessary metabolic processes for survival. Among eukaryotes, alternative oxidases have dispersed distribution and are found in plants, fungi, and protists, including Naegleria ssp. Naegleria species are free-living unicellular amoeboflagellates and include the pathogenic species of N. fowleri, the so-called "brain-eating amoeba." Using a multidisciplinary approach, we aimed to understand the evolution, localization, and function of AOX and the role that plays in Naegleria's biology. Our analyses suggest that AOX was present in last common ancestor of the genus and structure prediction showed that all functional residues are also present in Naegleria species. Using cellular and biochemical techniques, we also functionally characterize N. gruberi's AOX in its mitochondria, and we demonstrate that its inactivation affects its proliferation. Consequently, we discuss the benefits of the presence of this protein in Naegleria species, along with its potential pathogenicity role in N. fowleri. We predict that our findings will spearhead new explorations to understand the cell biology, metabolism, and evolution of Naegleria and other free-living relatives.

Cryptosporidium parvum is one of the major causes of neonatal calf diarrhoea resulting in reduced farm productivity and compromised animal welfare worldwide. Livestock act as a major reservoir of this parasite, which can be transmitted to humans directly and/or indirectly, posing a public health risk. Research reports on Cryptosporidium prevalence from the east Mediterranean region are scarce with even less originating from Cyprus. This study is the first to explore occurrence of Cryptosporidium spp. in young dairy calves on the island. A total of 242 faecal samples were collected from 10 dairy cattle farms in Cyprus, all of which were screened for Cryptosporidium spp. using nested-PCR amplification targeting the small subunit of the ribosomal RNA (18S rRNA) gene. The 60 kDa glycoprotein (gp60) gene was also sequenced for the samples identified as C. parvum positive to determine the subtypes present. Occurrence of Cryptosporidium was 43.8% (106/242) with at least one positive isolate in each farm sampled. Prevalence per farm ranged from 20¬‒64%, while C. bovis, C. ryanae and C. parvum were the only species identified. Amongst those, the latter was the predominant species, representing 51.8% of all positive samples. Four zoonotic subtypes were identified – IIaA14G1R1, IIaA15G1R1, IIaA15G2R1 and IIaA18G2R1. IIaA14G1R1 was the most abundant; representing 48.2% of all C. parvum positive samples and was also the most widespread. This is the first report of zoonotic subtypes of C. parvum circulating in Cyprus. These results highlight the need for further research into the parasite focusing on its diversity, prevalence, host-range and transmission dynamics on the island.

Cryptosporidium is comprised an apicomplexan parasitic protist, which infects a wide range of hosts, causing cryptosporidiosis. In cattle farms, the incidence of cryptosporidiosis results in high mortality in calves leading to considerable economic loss in the livestock industry. Infected animals may also act as a major reservoir of Cryptosporidium spp., in particular C. parvum, the most common cause of cryptosporidiosis in calves. This poses a significant risk to other farms via breeding centres, to trading of livestock and to human health. This study, funded by the Interreg-2-seas programme, is a part of a global project aimed at strategies to tackle cryptosporidiosis. To reach this target, it was essential to determine whether prevalence was dependent on the studied countries or if the issue was borderless. Indeed, C. parvum occurrence was assessed across dairy farms in certain regions of Belgium, France and the Netherlands. At the same time, the animal-to-animal transmission of the circulating C. parvum subtypes was studied. To accomplish this, 1084 faecal samples, corresponding to 57 dairy-farms from all three countries, were analysed. Well-established protocols amplifying the 18S rDNA and gp60 genes fragments, followed by DNA sequencing, were used for the detection and subtyping C. parvum; the DNA sequences obtained were further characterised using a combination of bioinformatics and phylogenetics methods. Our results show 25.7%, 24.9% and 20.8% prevalence of Cryptosporidium spp. in Belgium, France and the Netherlands respectively. Overall, 93% of the farms were Cryptosporidium positive. The gp60 subtyping demonstrated a significant number of the C. parvum positives belonged to the IIa allelic family, which has been also detected in humans. Consequently, this study highlights how widespread is C. parvum in dairy farms and endorses cattle as a major carrier of zoonotic C. parvum subtypes, which subsequently pose a significant threat to human health.

Blastocystis is the most common protist in the gut of humans and other animals having global distribution. Occasionally, this organism has also been reported in the environment. Transmission to humans occurs via the fecal-oral route, while water also comprises a transmission route. Blastocystis has been commonly found in rivers, lakes, and wells. Nonetheless, there is limited data about the prevalence and genetic diversity of Blastocystis in tap water. The main aim of this study was to examine the presence of Blastocystis subtypes in tap water (n=20) in a community in northern Thailand. Molecular characterization using the small subunit ribosomal RNA was used to screen for Blastocystis and identify the diversity of subtypes in samples. The overall prevalence was 30% with only subtype three (ST3) encountered in the tap water. These results indicate that tap water has a potential role in the transmission of this subtype in the studied community. Further investigations should focus on expanding sampling to include additional housing complexes and screening for Blastocystis in humans who are exposed to this water.

Introduction. Prototheca species are non-photosynthetic trebouxiophyte algae ubiquitously distributed in nature and can be found in sewage and soil. This microbial eukaryote causes human protothecosis in immunocompromised individuals. Thus, Prototheca presence in the stool of individuals without gastrointestinal symptoms has been reported only rarely. Hypothesis/Gap statement. There is an absence of detailed characterization of human Prototheca isolates. Aim. The aim of this study was to perform morphological and molecular characterization of Prototheca isolates obtained from human stool. Methodology. Prototheca was isolated from faecal samples of four individuals living in a rural area in Thailand. A combination of bioimaging along with molecular and bioinformatics tools was used to characterize the four strains. The growth rate was tested using four media and three temperature conditions. Phylogenetic analysis using the small subunit ribosomal RNA (SSU rRNA) and cytochrome b (cytb) was also performed. Results. Static and live microscopy demonstrated the various life stages of Prototheca and its major defining cellular characteristics. An optimized DNA extraction methodology that improves DNA yield is provided. Partial fragments of the SSU rRNA and cytb genes were obtained. Phylogenetic analysis placed all four strains in the clade with Prototheca bovis. More broadly, Prototheca was not monophyletic but split into at least two distinct clades instead. Conclusion. The results represent the first molecular characterization of Prototheca in Thailand. The study provides insight into transmission dynamics of the organism and potential caveats in estimating the global prevalence of Prototheca. These will spearhead further investigations on Prototheca occurrence in rural areas of both industrialized and developing nations.

Naegleria fowleri is both a pathogenic and a free-living microbial eukaryote, responsible for the development of primary amoebic meningoencephalitis (PAM) in humans. PAM is a rapid, severe and fatal underestimated infectious disease, which has been reported in countries with warmer climates. The major drawbacks with PAM are the lack of effective therapies and delay in diagnosis. The current frontline treatment presents a low rate of recovery (5%) and severe adverse effects. For example, many drug candidates lack efficacy, since they do not effectively cross the blood-brain-barrier. Consequently, more effective drugs are urgently needed. Herein, we report a new in vitro method suitable for medium- and high-throughput drug discovery assays, using the closely related Naegleria gruberi as a model. We have subsequently used this method to screen a library of 1175 Food and Drug Administration-approved drugs. As a result, we present three drugs (camptothecin, pyrimethamine, and terbinafine) that can be repurposed, and are anticipated to readily cross the blood-brain-barrier with activity against Naegleria species in therapeutically achievable concentrations. Successively, we integrated several in vitro assays that resulted in identifying fast-acting and high amoebicidal drugs. In conclusion, we present a new approach for the identification of anti-Naegleria drugs along with three potential drug candidates for further development for the treatment of PAM., Graphical abstract Image 1, Highlights • Novel in vitro approach for the identification of anti-Naegleria drugs • Modern drug discovery strategy by using a phenotypic screening against Naegleria • In vitro evaluation of 1175 FDA approved drugs against Naegleria • Camptothecin, pyrimethamine and terbinafine are promising drugs for the PAM • Higher activity, improved ADMET profile and bioavailability than amphotericin B

[Background] The opportunistic pathogen Naegleria fowleri establishes infection in the human brain, killing almost invariably within 2 weeks. The amoeba performs piece-meal ingestion, or trogocytosis, of brain material causing direct tissue damage and massive inflammation. The cellular basis distinguishing N. fowleri from other Naegleria species, which are all non-pathogenic, is not known. Yet, with the geographic range of N. fowleri advancing, potentially due to climate change, understanding how this pathogen invades and kills is both important and timely., [Results] Here, we report an -omics approach to understanding N. fowleri biology and infection at the system level. We sequenced two new strains of N. fowleri and performed a transcriptomic analysis of low- versus high-pathogenicity N. fowleri cultured in a mouse infection model. Comparative analysis provides an in-depth assessment of encoded protein complement between strains, finding high conservation. Molecular evolutionary analyses of multiple diverse cellular systems demonstrate that the N. fowleri genome encodes a similarly complete cellular repertoire to that found in free-living N. gruberi. From transcriptomics, neither stress responses nor traits conferred from lateral gene transfer are suggested as critical for pathogenicity. By contrast, cellular systems such as proteases, lysosomal machinery, and motility, together with metabolic reprogramming and novel N. fowleri proteins, are all implicated in facilitating pathogenicity within the host. Upregulation in mouse-passaged N. fowleri of genes associated with glutamate metabolism and ammonia transport suggests adaptation to available carbon sources in the central nervous system., [Conclusions] In-depth analysis of Naegleria genomes and transcriptomes provides a model of cellular systems involved in opportunistic pathogenicity, uncovering new angles to understanding the biology of a rare but highly fatal pathogen., EKH was supported by an AHFMR Fulltime Graduate Studentship and a Vanier Canada Graduate Scholarship. JBD is the Canada Research Chair in Evolutionary Cell Biology. Work in the Eliáš Lab is supported by CePaViP (CZ.02.1.01/0.0/0.0/16_019/0000759) and Czech Science Foundation project 18-18699S. This work was supported by CSIRO Land and Water (Australia).

Blastocystis is an opportunistic parasite commonly found in the intestines of humans and other animals. Despite its high prevalence, knowledge regarding Blastocystis biology within and outside the host is limited. Analysis of the metabolites produced by this anaerobe could provide insights that can help map its metabolism and determine its role in both health and disease. Due to its controversial pathogenicity, these metabolites could define its deterministic role in microbiome’s “health” and/or subsequently resolve Blastocystis’ potential impact in gastrointestinal health. A common method for elucidating the presence of these metabolites is through 1H nuclear magnetic resonance (NMR). However, there are currently no described benchmarked methods available to extract metabolites from Blastocystis for 1H NMR analysis. Herein, several extraction solvents, lysis methods and incubation temperatures were compared for their usefulness as an extraction protocol for this protozoan. Following extraction, the samples were freeze-dried, re-solubilized and analysed with 1H NMR. The results demonstrate that carrying out the procedure at room temperature using methanol as an extraction solvent and bead bashing as a lysis technique provides a consistent, reproducible and efficient method to extract metabolites from Blastocystis for NMR.

The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion., Bacteria use the type 2 secretion system to secrete enzymes and toxins across the outer membrane to the environment. Here the authors analyse the T2SS pathway in three protist lineages and suggest that the early mitochondrion may have been capable of secreting proteins into the cytosol.

Emerging infectious diseases are frequently zoonotic, often originating in wildlife, but enteric protozoa are considered relatively minor contributors. Opinions regarding whether pathogenic enteric protozoa may be transmitted between wildlife and humans have been shaped by our investigation tools, and have led to oscillations regarding whether particular species are zoonotic or have host-adapted life cycles. When the only approach for identifying enteric protozoa was morphology, it was assumed that many enteric protozoa colonized multiple hosts and were probably zoonotic. When molecular tools revealed genetic differences in morphologically identical species colonizing humans and other animals, host specificity seemed more likely. Parasites from animals found to be genetically identical - at the few genes investigated - to morphologically indistinguishable parasites from human hosts, were described as having zoonotic potential. More discriminatory molecular tools have now sub-divided some protozoa again. Meanwhile, some infection events indicate that, circumstances permitting, some “host-specific” protozoa, can actually infect various hosts. These repeated changes in our understanding are linked intrinsically to the investigative tools available. Here we review how molecular tools have assisted, or sometimes confused, our understanding of the public health threat from nine enteric protozoa and example wildlife hosts (Balantoides coli - wild boar; Blastocystis sp. - wild rodents; Cryptosporidium spp. - wild fish; Encephalitozoon spp. - wild birds; Entamoeba spp. - non-human primates; Enterocytozoon bieneusi - wild cervids; Giardia duodenalis - red foxes; Sarcocystis nesbitti - snakes; Toxoplasma gondii - bobcats). Molecular tools have provided evidence that some enteric protozoa in wildlife may infect humans, but due to limited discriminatory power, often only the zoonotic potential of the parasite is indicated. Molecular analyses, which should be as discriminatory as possible, are one, but not the only, component of the toolbox for investigating potential public health impacts from pathogenic enteric protozoa in wildlife., Graphical abstract Image 1, Highlights • Wildlife is a potential reservoir of disease agents that may infect humans. • The public health threat from enteric protozoa in wildlife is poorly understood. • Molecular tools may help in understanding this threat, but may also confuse. • We use nine enteric protozoa-wildlife host examples to review the current position. • Data accumulate, but more discriminatory tools and other approaches are important.

Blastocystis is a common and broadly distributed microbial eukaryote inhabiting the gut of humans and other animals. The genetic diversity of Blastocystis is extremely high comprising no less than 17 subtypes in mammals and birds. Nonetheless, little is known about the prevalence and distribution of Blastocystis subtypes colonising humans in Thailand. Molecular surveys of Blastocystis remain extremely limited and usually focus on the central, urban part of the country. To address this knowledge gap, we collected stool samples from a population of Thai adults (n = 178) residing in Chiang Rai Province. The barcoding region of the small subunit ribosomal RNA was employed to screen for Blastocystis and identify the subtype. Forty-one stool samples (23%) were identified as Blastocystis positive. Six of the nine subtypes that colonise humans were detected with subtype (ST) three being the most common (68%), followed by ST1 (17%) and ST7 (7%). Comparison of subtype prevalence across Thailand using all publicly available sequences showed that subtype distribution differs among geographic regions in the country. ST1 was most commonly encountered in the central region of Thailand, while ST3 dominated in the more rural north and northeast regions. ST2 was absent in the northeast, while ST7 was not found in the center. Thus, this study shows that ST prevalence and distribution differs not only among countries, but also among geographic regions within a country. Potential explanations for these observations are discussed herewith.

The main bacterial pathway for inserting proteins into the plasma membrane relies on the signal recognition particle (SRP), composed of the Ffh protein and an associated RNA component, and the SRP-docking protein FtsY. Eukaryotes use an equivalent system of archaeal origin to deliver proteins into the endoplasmic reticulum, whereas a bacteria-derived SRP and FtsY function in the plastid. Here we report on the presence of homologs of the bacterial Ffh and FtsY proteins in various unrelated plastid-lacking unicellular eukaryotes, namely Heterolobosea, Alveida, Goniomonas, and Hemimastigophora. The monophyly of novel eukaryotic Ffh and FtsY groups, predicted mitochondrial localization experimentally confirmed for Naegleria gruberi, and a strong alphaproteobacterial affinity of the Ffh group, collectively suggest that they constitute parts of an ancestral mitochondrial signal peptide-based protein-targeting system inherited from the last eukaryotic common ancestor, but lost from the majority of extant eukaryotes. The ability of putative signal peptides, predicted in a subset of mitochondrial-encoded N. gruberi proteins, to target a reporter fluorescent protein into the endoplasmic reticulum of Trypanosoma brucei, likely through their interaction with the cytosolic SRP, provided further support for this notion. We also illustrate that known mitochondrial ribosome-interacting proteins implicated in membrane protein targeting in opisthokonts (Mba1, Mdm38, and Mrx15) are broadly conserved in eukaryotes and nonredundant with the mitochondrial SRP system. Finally, we identified a novel mitochondrial protein (MAP67) present in diverse eukaryotes and related to the signal peptide-binding domain of Ffh, which may well be a hitherto unrecognized component of the mitochondrial membrane protein-targeting machinery.

Of the 40 describedNaegleriaspecies, onlyN. fowlerican establish infection in humans, killing almost invariably within two weeks. In the brain, the amoeba performs piece-meal ingestion, or trogocytosis, of brain material causing massive inflammation. Conversely, its close relativeNaegleria gruberi, which is used as a laboratory model organism, is non-pathogenic. The exact pathogenicity factors distinguishingN. fowlerifrom its harmless relatives are unclear. We have here taken an -omics approach to understandingN. fowleribiology and infection at the system level. We provide the first analysis of genomic diversity between strains, finding little conservation in synteny but high conservation in protein complement. We also demonstrate that theN. fowlerigenome encodes a similarly complete cellular repertoire to that found inN. gruberi. Our comparative genomic analysis, together with a transcriptomic analysis of low versus high pathogenicityN. fowlericultured in a mouse infection model, allowed us to construct a model of cellular systems involved in pathogenicity and furthermore provides ~500 novel candidate pathogenicity factors in this currently rare but highly fatal pathogen.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways., This collaborative effort was supported by the Gordon and Betty Moore Foundation EMS Program of the Marine Microbiology Initiative (grant nos. GBMF4972 and 4972.01 to F.-Y.B.; GBMF4970 and 4970.01 to M.A. and A.Z.W.; GBMF3788 to A.Z.W.; GBMF 4968 and 4968.01 to H.C.; GBMF4984 to V.H.; GBMF4974 and 4974.01 to C. Brownlee; GBMF4964 to Y. Hirakawa; GBMF4961 to T. Mock; GBMF4958 to P.S.; GBMF4957 to A.T.; GBMF4960 to G.J.S.; GBMF4979 to K.C.; GBMF4982 and 4982.01 to J.L.C.; GBMF4964 to P.J.K.; GBMF4981 to P.v.D.; GBMF5006 to A.E.A.; GBMF4986 to C.M.; GBMF4962 to J.A.F.R.; GBMF4980 and 4980.01 to S.L.; GBMF 4977 and 4977.01 to R.F.W.; GBMF4962.01 to C.H.S.; GBMF4985 to J.M.; GBMF4976 and 4976.01 to C.H.; GBMF4963 and 4963.01 to V.E.; GBMF5007 to C.L.D.; GBMF4983 and 4983.01 to J.L.; GBMF4975 and 4975.01 to A.D.T.; GBMF4973 and 4973.01 to I.R.-T. and GBMF4965 to N.K.), by The Leverhulme Trust (RPG-2017-364) to T. Mock and A. Hopes, and by ERD funds (16_019/0000759) from the Czech Ministry of Education to J.L.

Blastocystis is a microbial eukaryote, considered to be the most prevalent microbe colonizing the human gut, colonising approximately one billion individuals worldwide. Although Blastocystis presence has been linked to intestinal disorders, its pathogenicity still remains controversial due to its high prevalence in asymptomatic carriers. Having 17 genetic subtypes, Blastocystis is extremely diverse and can withstand fluctuations of oxygen in the gut. Blastocystis harbors peculiar mitochondrion-related organelles (MROs), which are considered to be an intermediate form between a typical aerobic mitochondrion and an obligate anaerobic hydrogenosome. Another interesting fact about Blastocystis concerns its mixed genome: 2.5% of the Blastocystis proteins have been laterally acquired from eukaryotes and prokaryotes. These acquired genes are associated with carbohydrate scavenging and metabolism, anaerobic amino acid and nitrogen metabolism, oxygen-stress resistance, and pH homeostasis. In addition, Blastocystis has proteins associated with secretion that are potentially involved in infection, escaping host defense and even affect composition of the prokaryotic microbiome and inflammation of the gut. In this chapter, we will challenge the state-of-the-art on Blastocystis knowledge, and we will present published data that can be used to understand the genomic adaptations of this microbial organism and its role within the microbiome of the hosts.

Simple Summary The last decade has seen a large increase into research on the microbiome and its roles in health and disease. The majority of this work has focused primarily on the bacterial component of the microbiome. However, there is evidence to suggest that microbial eukaryotes colonising the gastrointestinal tract may have roles in the shaping and structuring of the microbiota and are thus likely to influence disease outcomes and host health. The aim of this study was to investigate the questionable pathogen Blastocystis and expand the number of studies on non-primate hosts, which address its associations with bacterial communities in the gut. Herein we examined the bacterial gut microbiota of Blastocystis positive and negative water voles. Results demonstrate no association of Blastocystis, bacterial richness and community composition. Nonetheless, the abundance of some taxa was affected in Blastocystis positive samples. The lack of significant shifts in community abundance between Blastocystis carriers and non-carriers indicates that this microbe may not be having a profound impact on bacterial communities in the gut of these animals. Abstract (1) Background: Blastocystis is a microbial eukaryote inhabiting the gastrointestinal tract of a broad range of animals including humans. Several studies have shown that the organism is associated with specific microbial profiles and bacterial taxa that have been deemed beneficial to intestinal and overall health. Nonetheless, these studies are focused almost exclusively on humans, while there is no similar information on other animals. (2) Methods: Using a combination of conventional PCR, cloning and sequencing, we investigated presence of Blastocystis along with Giardia and Cryptosporidium in 16 captive water voles sampled twice from a wildlife park. We also characterised their bacterial gut communities. (3) Results: Overall, alpha and beta diversities between water voles with and without Blastocystis did not differ significantly. Differences were noted only on individual taxa with Treponema and Kineothrix being significantly reduced in Blastocystis positive water voles. Grouping according to antiprotozoal treatment and presence of other protists did not reveal any differences in the bacterial community composition either. (4) Conclusion: Unlike human investigations, Blastocystis does not seem to be associated with specific gut microbial profiles in water voles.

Maps and nucleotide sequences of the Naegleria gruberi transfection plasmids (pNaeg-HYG and pNaeg-NEO) reported in Faktorová D. et al.

Blastocystis is an anaerobic microbial eukaryote that inhabits the gut of various animals. While it was considered to be a parasite, its pathogenicity is controversial, since recent estimations suggest that Blastocystis could be present in more than one billion humans. Blastocystis belongs to the stramenopiles (heterokonts), a diverse group of eukaryotes that also include unicellular diatoms and giant multicellular kelps. The Blastocystis cell lacks typical features of other stramenopiles; its genome has laterally acquired many genes from other prokaryotes and eukaryotes, resulting in having exceptional biochemistry and unique adaptations of its mitochondria. For example, Blastocystis mitochondrion-related organelles (MROs) have characteristics of typical mitochondria, including Complexes I and II from electron transport chain, mitochondrial DNA, Fe-S cluster assembly and amino acid metabolism, but also proteins that are typically present in obligate anaerobes including FeFe-hydrogenase, pyruvate metabolism and alternative oxidase. Some of the pathways have been localized and biochemically characterized, providing a better understanding on the functions of these organelles. In this chapter, we will present and examine the status quo regarding the biology and morphology of Blastocystis MROs, and we will discuss future avenues on exploring even further adaptations of these organelles.

Gregarine apicomplexans are closely related to parasites such as Plasmodium, Toxoplasma, and Cryptosporidium, which are causing severe health and economic burdens. Colonizing only invertebrates and having no obvious medical relevance, they are mostly ignored in ‘omics’ studies, although gregarines are the most basal apicomplexans and therefore key players in the understanding of the evolution of parasitism in the Apicomplexa from free-living ancestors. They belong to the only exclusively parasitic phylum of eukaryotes, but is this perception actually true? The effects of gregarines on their hosts seem to cover the whole spectrum of symbiosis from mutualistic to parasitic. We suggest future research directions to understand the evolutionary role of gregarines, by elucidating their biology and interaction with their hosts and the hosts’ microbiota.

Background Cryptosporidium is an important gut microbe whose contributions towards infant and immunocompromise patient mortality rates are steadily increasing. Over the last decade, we have seen the development of various tools and methods for studying Cryptosporidium infection and its interactions with their hosts. One area that is sorely overlooked is the effect infection has on host metabolic processes. Results Using a 1H nuclear magnetic resonance approach to metabolomics, we have explored the nature of the mouse gut metabolome as well as providing the first insight into the metabolome of an infected cell line. Statistical analysis and predictive modelling demonstrated new understandings of the effects of a Cryptosporidium infection, while verifying the presence of known metabolic changes. Of note is the potential contribution of host derived taurine to the diarrhoeal aspects of the disease previously attributed to a solely parasite-based alteration of the gut environment, in addition to other metabolites involved with host cell catabolism. Conclusion This approach will spearhead our understanding of the Cryptosporidium-host metabolic exchange and provide novel targets for tackling this deadly parasite. Electronic supplementary material The online version of this article (10.1186/s13099-019-0293-x) contains supplementary material, which is available to authorized users.