Microbiome diversity is a modifiable virulence factor for cryptosporidiosis.

Cryptosporidium spp. infection causes significant disease in immunosuppressed individuals and children under the age of 5 years. The severity of the pathological presentation of cryptosporidiosis is a function of the host and parasite genotypes, host immune status, and the enteric environment or microbiome of the host. Cryptosporidiosis often presents with abdominal pain and severe diarrhoea and is associated with intestinal dysbiosis and inflammation. Our systematic analysis of the available literature revealed that bacterial diversity is reduced during infection in larger animal models, lending support to recent studies which indicate that the use of probiotics or the presence of a naturally diverse gut microbiome can prevent or minimise pathology caused by gastrointestinal pathogens. In summary, we present evidence that the presence of a diverse gut microbiome, natural or induced, reduces both symptomatic pathology and oocyst output.

American variants of Cryptosporidium hominis: Over-sexed and over here?

A new study in this issue of Cell Host & Microbe from Huang et al. provides important insights into the global epidemiology of human-infectious Cryptosporidium and mechanisms leading to the rapid emergence and rise to dominance of new, possibly more virulent, parasite strains.

Euglena International Network (EIN): Driving euglenoid biotechnology for the benefit of a challenged world.

Euglenoids (Euglenida) are unicellular flagellates possessing exceptionally wide geographical and ecological distribution. Euglenoids combine a biotechnological potential with a unique position in the eukaryotic tree of life. In large part these microbes owe this success to diverse genetics including secondary endosymbiosis and likely additional sources of genes. Multiple euglenoid species have translational applications and show great promise in production of biofuels, nutraceuticals, bioremediation, cancer treatments and more exotically as robotics design simulators. An absence of reference genomes currently limits these applications, including development of efficient tools for identification of critical factors in regulation, growth or optimization of metabolic pathways. The Euglena International Network (EIN) seeks to provide a forum to overcome these challenges. EIN has agreed specific goals, mobilized scientists, established a clear roadmap (Grand Challenges), connected academic and industry stakeholders and is currently formulating policy and partnership principles to propel these efforts in a coordinated and efficient manner.

Genomic erosion in a demographically recovered bird species during conservation rescue.

The pink pigeon (Nesoenas mayeri) is an endemic species of Mauritius that has made a remarkable recovery after a severe population bottleneck in the 1970s to early 1990s. Prior to this bottleneck, an ex situ population was established from which captive-bred individuals were released into free-living subpopulations to increase population size and genetic variation. This conservation rescue led to rapid population recovery to 400-480 individuals, and the species was twice downlisted on the International Union for the Conservation of Nature (IUCN) Red List. We analyzed the impacts of the bottleneck and genetic rescue on neutral genetic variation during and after population recovery (1993-2008) with restriction site-associated sequencing, microsatellite analyses, and quantitative genetic analysis of studbook data of 1112 birds from zoos in Europe and the United States. We used computer simulations to study the predicted changes in genetic variation and population viability from the past into the future. Genetic variation declined rapidly, despite the population rebound, and the effective population size was approximately an order of magnitude smaller than census size. The species carried a high genetic load of circa 15 lethal equivalents for longevity. Our computer simulations predicted continued inbreeding will likely result in increased expression of deleterious mutations (i.e., a high realized load) and severe inbreeding depression. Without continued conservation actions, it is likely that the pink pigeon will go extinct in the wild within 100 years. Conservation rescue of the pink pigeon has been instrumental in the recovery of the free-living population. However, further genetic rescue with captive-bred birds from zoos is required to recover lost variation, reduce expression of harmful deleterious variation, and prevent extinction. The use of genomics and modeling data can inform IUCN assessments of the viability and extinction risk of species, and it helps in assessments of the conservation dependency of populations.

La paloma rosada (Nesoenas mayeri) es una especie endémica de Mauricio que se ha recuperado impresionantemente después de un grave cuello de botella poblacional a principios de la década de 1970 que duró hasta inicios de la década de 1990. Antes de este cuello de botella se había establecido una población ex situ de la cual se liberaban individuos reproducidos en cautiverio a las subpoblaciones en libertad para incrementar la variación genética y el tamaño poblacional. Este rescate de conservación derivó en una recuperación rápida de la población (400-480 individuos) y la especie cambió positivamente de categoría dos veces en la Lista Roja de la Unión Internacional para la Conservación de la Naturaleza (UICN). Analizamos los impactos del cuello de botella y el rescate genético sobre la variación genética neutral durante y después de la recuperación poblacional (de 1993 a 2008) mediante secuenciación RAD, análisis de microsatélites y análisis genéticos cuantitativos de los datos del libro genealógico de 1112 aves ubicadas en zoológicos de Europa y los Estados Unidos. Usamos simulaciones por computadora para estudiar los cambios pronosticados en la variación genética y en la viabilidad poblacional del pasado hacia el futuro. La variación genética declinó rápidamente, a pesar de la recuperación poblacional, y el tamaño efectivo de la población fue aproximadamente un orden de magnitud más pequeño que el tamaño del censo. La especie contó con una carga genética elevada de casi 15 equivalentes letales para la longevidad. Nuestras simulaciones pronostican que la endogamia continua probablemente resultará en un incremento en la expresión de mutaciones deletéreas (es decir, una carga realizada elevada) y en una depresión endogámica severa. Sin acciones continuas para la conservación, es probable que la paloma rosada esté extinta en vida libre dentro de cien años. El rescate de conservación de la paloma rosada ha sido fundamental en la recuperación de la población silvestre; sin embargo, se requiere de un rescate genético adicional con las aves de reproducción en cautiverio de los zoológicos para recuperar la variación perdida, reducir la expresión de la variación deletérea dañina y prevenir la extinción. El uso de la genómica y los datos modelados puede orientar las valoraciones de la UICN sobre la viabilidad y el riesgo de extinción de las especies, además de que ayuda en la evaluación de la dependencia que tienen las poblaciones de la conservación.

Global Population Genomics of Two Subspecies of Cryptosporidium hominis during 500 Years of Evolution.

Cryptosporidiosis is a major global health problem and a primary cause of diarrhea, particularly in young children in low- and middle-income countries (LMICs). The zoonotic Cryptosporidium parvum and anthroponotic Cryptosporidium hominis cause most human infections. Here, we present a comprehensive whole-genome study of C. hominis, comprising 114 isolates from 16 countries within five continents. We detect two lineages with distinct biology and demography, which diverged circa 500 years ago. We consider these lineages two subspecies and propose the names C. hominis hominis and C. hominis aquapotentis (gp60 subtype IbA10G2). In our study, C. h. hominis is almost exclusively represented by isolates from LMICs in Africa and Asia and appears to have undergone recent population contraction. In contrast, C. h. aquapotentis was found in high-income countries, mainly in Europe, North America, and Oceania, and appears to be expanding. Notably, C. h. aquapotentis is associated with high rates of direct human-to-human transmission, which may explain its success in countries with well-developed environmental sanitation infrastructure. Intriguingly, we detected genomic regions of introgression following secondary contact between the subspecies. This resulted in high diversity and divergence in genomic islands of putative virulence genes, including muc5 (CHUDEA2_430) and a hypothetical protein (CHUDEA6_5270). This diversity is maintained by balancing selection, suggesting a co-evolutionary arms race with the host. Finally, we find that recent gene flow from C. h. aquapotentis to C. h. hominis, likely associated with increased human migration, maybe driving the evolution of more virulent C. hominis variants.

Parasite-Mediated Remodeling of the Host Microfilament Cytoskeleton Enables Rapid Egress of Trypanosoma cruzi following Membrane Rupture.

Chagas' disease arises as a direct consequence of the lytic cycle of Trypanosoma cruzi in the mammalian host. While invasion is well studied for this pathogen, study of egress has been largely neglected. Here, we provide the first description of T. cruzi egress documenting a coordinated mechanism by which T. cruzi engineers its escape from host cells in which it has proliferated and which is essential for maintenance of infection and pathogenesis. Our results indicate that this parasite egress is a sudden event involving coordinated remodeling of host cell cytoskeleton and subsequent rupture of host cell plasma membrane. We document that host cells maintain plasma membrane integrity until immediately prior to parasite release and report the sequential transformation of the host cell's actin cytoskeleton from normal meshwork in noninfected cells to spheroidal cages-a process initiated shortly after amastigogenesis. Quantification revealed gradual reduction in F-actin over the course of infection, and using cytoskeletal preparations and electron microscopy, we were able to observe disruption of the F-actin proximal to intracellular trypomastigotes. Finally, Western blotting experiments suggest actin degradation driven by parasite proteases, suggesting that degradation of cytoskeleton is a principal component controlling the initiation of egress. Our results provide the first description of the cellular mechanism that regulates the lytic component of the T. cruzi lytic cycle. We show graphically how it is possible to preserve the envelope of host cell plasma membrane during intracellular proliferation of the parasite and how, in cells packed with amastigotes, differentiation into trypomastigotes may trigger sudden egress. IMPORTANCE Understanding how Trypanosoma cruzi interacts with host cells has been transformed by high-quality studies that have examined in detail the mechanisms of T. cruzi host cell invasion. In contrast, little is known about the latter stages of the parasite's lytic cycle: how parasites egress and thereby sustain round after round of infection. Our results show that once in the host cell cytosol and having undergone amastigogenesis, T. cruzi begins to alter the host cell cytoskeleton, remodeling normal F-actin meshworks into encapsulating spheroidal cages. Filamentous actin diminishes over the course of the lytic cycle, and just prior to egress, the filaments comprising the cages are severely degraded where adjacent to the parasites. We conclude that sudden egress follows breach of the containment afforded by the actin cytoskeleton and subsequent plasma membrane rupture-a process that when understood in molecular detail may serve as a target for future novel therapeutic interventions.

Decolonising Parasitology: The Case of Trypanosoma brucei rhodesiense.

Trypanosoma brucei rhodesiense was named after Rhodesia which, in turn, was named after the British imperialist and white supremacist Cecil Rhodes. In the light of the Black Lives Matter movement and contemporary consciousness of postcolonial legacy, it seems opportune to reconsider the subspecies name. Pros and cons of renaming T. b. rhodesiense are discussed.

Trypanocidal and leishmanicidal activity of six limonoids.

Six limonoids [kotschyienone A and B (1, 2), 7-deacetylgedunin (3), 7-deacetyl-7-oxogedunin (4), andirobin (5) and methyl angolensate (6)] were investigated for their trypanocidal and leishmanicidal activities using bloodstream forms of Trypanosoma brucei and promastigotes of Leishmania major. Whereas all compounds showed anti-trypanosomal activity, only compounds 1-4 displayed anti-leishmanial activity. The 50% growth inhibition (GI50) values for the trypanocidal and leishmanicidal activity of the compounds ranged between 2.5 and 14.9 µM. Kotschyienone A (1) was found to be the most active compound with a minimal inhibition concentration (MIC) value of 10 µM and GI50 values between 2.5 and 2.9 µM. Only compounds 1 and 3 showed moderate cytotoxicity against HL-60 cells with MIC and GI50 values of 100 µM and 31.5-46.2 µM, respectively. Compound 1 was also found to show activity against intracellular amastigotes of L. major with a GI50 value of 1.5 µM. The results suggest that limonoids have potential as drug candidates for the development of new treatments against trypanosomiasis and leishmaniasis.

Improved subtyping affords better discrimination of Trichomonas gallinae strains and suggests hybrid lineages.

Trichomonas gallinae is a protozoan pathogen that causes avian trichomonosis typically associated with columbids (canker) and birds of prey (frounce) that predate on them, and has recently emerged as an important cause of passerine disease. An archived panel of DNA from North American (USA) birds used initially to establish the ITS ribotypes was reanalysed using Iron hydrogenase (FeHyd) gene sequences to provide an alphanumeric subtyping scheme with improved resolution for strain discrimination. Thirteen novel subtypes of T. gallinae using FeHyd gene as the subtyping locus are described. Although the phylogenetic topologies derived from each single marker are complementary, they are not entirely congruent. This may reflect the complex genetic histories of the isolates analysed which appear to contain two major lineages and several that are hybrid. This new analysis consolidates much of the phylogenetic signal generated from the ITS ribotype and provides additional resolution for discrimination of T. gallinae strains. The single copy FeHyd gene provides higher resolution genotyping than ITS ribotype alone. It should be used where possible as an additional, single-marker subtyping tool for cultured isolates.

Characterization of Evolutionarily Conserved Trypanosoma cruzi NatC and NatA-N-Terminal Acetyltransferase Complexes.

Protein N-terminal acetylation is a co- and posttranslational modification, conserved among eukaryotes. It determines the functional fate of many proteins including their stability, complex formation, and subcellular localization. N-terminal acetyltransferases (NATs) transfer an acetyl group to the N-termini of proteins, and the major NATs in yeast and humans are NatA, NatB, and NatC. In this study, we characterized the Trypanosoma cruzi (T. cruzi) NatC and NatA protein complexes, each consisting of one catalytic subunit and predicted auxiliary subunits. The proteins were found to be expressed in the three main life cycle stages of the parasite, formed stable complexes in vivo, and partially cosedimented with the ribosome in agreement with a cotranslational function. An in vitro acetylation assay clearly demonstrated that the acetylated substrates of the NatC catalytic subunit from T. cruzi were similar to those of yeast and human NatC, suggesting evolutionary conservation of function. An RNAi knockdown of the Trypanosoma brucei (T. brucei) NatC catalytic subunit indicated that reduced NatC-mediated N-terminal acetylation of target proteins reduces parasite growth.

Evolutionary genomics of anthroponosis in Cryptosporidium.

Human cryptosporidiosis is the leading protozoan cause of diarrhoeal mortality worldwide, and a preponderance of infections is caused by Cryptosporidium hominis and C. parvum. Both species consist of several subtypes with distinct geographical distributions and host preferences (that is, generalist zoonotic and specialist anthroponotic subtypes). The evolutionary processes that drive the adaptation to the human host and the population structures of Cryptosporidium remain unknown. In this study, we analyse 21 whole-genome sequences to elucidate the evolution of anthroponosis. We show that Cryptosporidium parvum splits into two subclades and that the specialist anthroponotic subtype IIc-a shares a subset of loci with C. hominis that is undergoing rapid convergent evolution driven by positive selection. C. parvum subtype IIc-a also has an elevated level of insertion and deletion mutations in the peri-telomeric genes, which is also a characteristic of other specialist subtypes. Genetic exchange between Cryptosporidium subtypes plays a prominent role throughout the evolution of the genus. Interestingly, recombinant regions are enriched for positively selected genes and potential virulence factors, which indicates adaptive introgression. Analysis of 467 gp60 sequences collected from locations across the world shows that the population genetic structure differs markedly between the main zoonotic subtype (isolation-by-distance) and the anthroponotic subtype (admixed population structure). We also show that introgression between the four anthroponotic Cryptosporidium subtypes and species included in this study has occurred recently, probably within the past millennium.

Anthroponotic transmission of Cryptosporidium parvum predominates in countries with poorer sanitation: a systematic review and meta-analysis.

BACKGROUND: Globally cryptosporidiosis is one of the commonest causes of mortality in children under 24 months old and may be associated with important longterm health effects. Whilst most strains of Cryptosporidium parvum are zoonotic, C. parvum IIc is almost certainly anthroponotic. The global distribution of this potentially important emerging infection is not clear. METHODS: We conducted a systematic review of papers identifying the subtype distribution of C. parvum infections globally. We searched PubMed and Scopus using the following key terms Cryptospor* AND parvum AND (genotyp* OR subtyp* OR gp60). Studies were eligible for inclusion if they had found C. parvum within their human study population and had subtyped some or all of these samples using standard gp60 subtyping. Pooled analyses of the proportion of strains being of the IIc subtype were determined using StatsDirect. Meta-regression analyses were run to determine any association between the relative prevalence of IIc and Gross Domestic Product, proportion of the population with access to improved drinking water and improved sanitation. RESULTS: From an initial 843 studies, 85 were included in further analysis. Cryptosporidium parvum IIc was found in 43 of these 85 studies. Across all studies the pooled estimate of relative prevalence of IIc was 19.0% (95% CI: 12.9-25.9%), but there was substantial heterogeneity. In a meta-regression analysis, the relative proportion of all C. parvum infections being IIc decreased as the percentage of the population with access to improved sanitation increased and was some 3.4 times higher in those studies focussing on HIV-positive indivduals. CONCLUSIONS: The anthroponotic C. parvum IIc predominates primarily in lower-income countries with poor sanitation and in HIV-positive individuals. Given the apparent enhanced post-infectious virulence of the other main anthroponotic species of Cryptosporidium (C. hominis), it is important to learn about the impact of this subtype on human health.

Giardia secretome highlights secreted tenascins as a key component of pathogenesis.

Background: Giardia is a protozoan parasite of public health relevance that causes gastroenteritis in a wide range of hosts. Two genetically distinct lineages (assemblages A and B) are responsible for the human disease. Although it is clear that differences in virulence occur, the pathogenesis and virulence of Giardia remain poorly understood. Results: The genome of Giardia is believed to contain open reading frames that could encode as many as 6000 proteins. By successfully applying quantitative proteomic analyses to the whole parasite and to the supernatants derived from parasite culture of assemblages A and B, we confirm expression of ∼1600 proteins from each assemblage, the vast majority of which are common to both lineages. To look for signature enrichment of secreted proteins, we considered the ratio of proteins in the supernatant compared with the pellet, which defined a small group of enriched proteins, putatively secreted at a steady state by cultured growing trophozoites of both assemblages. This secretome is enriched with proteins annotated to have N-terminal signal peptide. The most abundant secreted proteins include known virulence factors such as cathepsin B cysteine proteases and members of a Giardia superfamily of cysteine-rich proteins that comprise variant surface proteins, high-cysteine membrane proteins, and a new class of virulence factors, the Giardia tenascins. We demonstrate that physiological function of human enteric epithelial cells is disrupted by such soluble factors even in the absence of the trophozoites. Conclusions: We are able to propose a straightforward model of Giardia pathogenesis incorporating key roles for the major Giardia-derived soluble mediators.

Evaluation of marking of peer marking in oral presentation.

BACKGROUND: Peer marking is an important skill for students, helping them to understand the process of learning and assessment. This method is increasingly used in medical education, particularly in formative assessment. However, the use of peer marking in summative assessment is not widely adopted because many teachers are concerned about biased marking by students of their peers. OBJECTIVE: The aim of this study was to investigate whether marking of summative peer assessment can improve the reliability of peer marking. METHODS: In a retrospective analysis, the peer-marking results of a summative assessment of oral presentations of two cohorts of students were compared. One group of students was told that their peer marks would be assessed against a benchmark consisting of the average of examiner marks and that these scores together with the peer and examiner marks would form their final exam results. The other group of students were just informed that their final exam results would be determined based on the examiner and peer marks. RESULTS: Based on examiner marks, both groups of students performed similarly in their summative assessment, agreement between student markers was less consistent and more polar than the examiners. When compared with the examiners, students who were told that their peer marking would be scored were more generous markers (their average peer mark was 2.4 % points higher than the average examiner mark) while students who were not being scored on their marking were rather harsh markers (their average peer mark was 4.2 % points lower than the average examiner mark), with scoring of the top-performing students most affected. CONCLUSIONS: Marking of peer marking had a small effect on the marking conduct of students in summative assessment of oral presentation but possibly indicated a more balanced marking performance.

Molecular characterization of Trichomonas gallinae isolates recovered from the Canadian Maritime provinces' wild avifauna reveals the presence of the genotype responsible for the European finch trichomonosis epidemic and additional strains.

Finch trichomonosis, caused by Trichomonas gallinae, emerged in the Canadian Maritime provinces in 2007 and has since caused ongoing mortality in regional purple finch (Carpodacus purpureus) and American goldfinch (Carduelis tristis) populations. Trichomonas gallinae was isolated from (1) finches and rock pigeons (Columbia livia) submitted for post-mortem or live-captured at bird feeding sites experiencing trichomonosis mortality; (2) bird seed at these same sites; and (3) rock pigeons live-captured at known roosts or humanely killed. Isolates were characterized using internal transcribed spacer (ITS) region and iron hydrogenase (Fe-hyd) gene sequences. Two distinct ITS types were found. Type A was identical to the UK finch epidemic strain and was isolated from finches and a rock pigeon with trichomonosis; apparently healthy rock pigeons and finches; and bird seed at an outbreak site. Type B was obtained from apparently healthy rock pigeons. Fe-hyd sequencing revealed six distinct subtypes. The predominant subtype in both finches and the rock pigeon with trichomonosis was identical to the UK finch epidemic strain A1. Single nucleotide polymorphisms in Fe-hyd sequences suggest there is fine-scale variation amongst isolates and that finch trichomonosis emergence in this region may not have been caused by a single spill-over event.

The finch epidemic strain of Trichomonas gallinae is predominant in British non-passerines.

Avian trichomonosis, caused by the flagellated protozoan Trichomonas gallinae, is a recently emerged infectious disease of British passerines. The aetiological agent, a clonal epidemic strain of the parasite, has caused unprecedented finch mortality and population-level declines in Britain and has since spread to continental Europe. To better understand the potential origin of this epidemic and to further investigate its host range, T. gallinae DNA extracts were collected from parasite culture and tissue samples from a range of avian species in Britain. Sequence typing at the ITS1/5.8S rRNA/ITS2 region resolved three distinct ITS region types circulating in free-ranging British birds. Subtyping by sequence analyses at the Fe-hydrogenase gene demonstrated further strain variation within these ITS region types. The UK finch epidemic strain was preponderant amongst columbids sampled, however, wide strain diversity was encountered in isolates from a relatively small number of pigeons, suggesting further strains present in columbid populations across the UK are yet to be identified. Fe-hydrogenase gene sequence data in isolates from birds of prey with disease were predominantly identical to the UK finch epidemic strain, demonstrating its presence as a virulent strain in UK birds of prey since at least 2009.

Trans-sialidase stimulates eat me response from epithelial cells.

Epithelial cell invasion by the protozoan parasite Trypanosoma cruzi is enhanced by the presence of an enzyme expressed on its cell surface during the trypomastigote life cycle stage. The enzyme, trans-sialidase (TS), is a member of one of the largest gene families expressed by the parasite and the role of its activity in mediating epithelial cell entry has not hitherto been understood. Here we show that the T. cruzi TS generates an eat me signal which is capable of enabling epithelial cell entry. We have utilized purified, recombinant, active (TcTS) and inactive (TcTS2V0) TS coated onto beads to challenge an epithelial cell line. We find that TS activity acts upon G protein coupled receptors present at the epithelial cell synapse with the coated bead, thereby enhancing cell entry. By so doing, we provide evidence that TS proteins bind glycans, mediate the formation of distinct synaptic domains and promote macropinocytotic uptake of microparticles into a perinuclear compartment in a manner which may emulate entosis.