Mass mortality event of round sardinella Sardinella aurita Valenciennes associated with Glugea Thélohan, 1891 microsporidian infection off the southern Italian coast.

Intracellular parasites of the genus Glugea Thélohan, 1891 (Microsporidia) comprise about 34 putative species capable of causing high morbidity and mortality in freshwater and marine teleost fishes. In this study, we report on the first mass mortality event associated with Glugea sp. infecting free-ranging round sardinella Sardinella aurita in the southern Tyrrhenian Sea (Italy). Here, we describe the ultrastructure of mature spores of this microsporidian and characterize it molecularly, as well as report its phylogenetic position. Most of the affected fish showed an irregular swelling of its abdomen. At necropsy, a variable number of xenomas, spherical to ellipsoidal in shape, were found in the peritoneal cavity strongly attached to the viscera of all fish. Histological analysis revealed varying severity of chronic inflammation along with occasional necrosis in visceral organs associated with multiple xenoma proliferation. These pathological findings were considered the main cause of this mass mortality event. Morphologically, the present material was closely related to G. sardinellesis and G. thunni. The phylogenetically closest taxa to the newly SSU rDNA sequence were G. thunni and an erroneusly identified  G. plecoglossi, which were very closely related to each other, also suggesting that all these sequences might belong to the same species.

FiCRoN, a deep learning-based algorithm for the automatic determination of intracellular parasite burden from fluorescence microscopy images.

Protozoan parasites are responsible for dramatic, neglected diseases. The automatic determination of intracellular parasite burden from fluorescence microscopy images is a challenging problem. Recent advances in deep learning are transforming this process, however, high-performance algorithms have not been developed. The limitations in image acquisition, especially for intracellular parasites, make this process complex. For this reason, traditional image-processing methods are not easily transferred between different datasets and segmentation-based strategies do not have a high performance. Here, we propose a novel method FiCRoN, based on fully convolutional regression networks (FCRNs), as a promising new tool for estimating intracellular parasite burden. This estimation requires three values, intracellular parasites, infected cells and uninfected cells. FiCRoN solves this problem as multi-task learning: counting by regression at two scales, a smaller one for intracellular parasites and a larger one for host cells. It does not use segmentation or detection, resulting in a higher generalization of counting tasks and, therefore, a decrease in error propagation. Linear regression reveals an excellent correlation coefficient between manual and automatic methods. FiCRoN is an innovative freedom-respecting image analysis software based on deep learning, designed to provide a fast and accurate quantification of parasite burden, also potentially useful as a single-cell counter.

Intra-host Trypanosoma cruzi strain dynamics shape disease progression: the missing link in Chagas disease pathogenesis.

Chronic Chagasic cardiomyopathy develops years after infection in 20-40% of patients, but disease progression is poorly understood. Here, we assessed Trypanosoma cruzi parasite dynamics and pathogenesis over a 2.5-year period in naturally infected rhesus macaques. Individuals with better control of parasitemia were infected with a greater diversity of parasite strains compared to those with increasing parasitemia over time. Also, the in vivo parasite multiplication rate decreased with increasing parasite diversity, suggesting competition among strains or a stronger immune response in multiple infections. Significant differences in electrocardiographic (ECG) profiles were observed in Chagasic macaques compared to uninfected controls, suggesting early conduction defects, and changes in ECG patterns over time were observed only in macaques with increasing parasitemia and lower parasite diversity. Disease progression was also associated with plasma fibronectin degradation, which may serve as a biomarker. These data provide a novel framework for the understanding of Chagas disease pathogenesis, with parasite diversity shaping disease progression.IMPORTANCEChagas disease progression remains poorly understood, and patients at increased risk of developing severe cardiac disease cannot be distinguished from those who may remain asymptomatic. Monitoring of Trypanosoma cruzi strain dynamics and pathogenesis over 2-3 years in naturally infected macaques shows that increasing parasite diversity in hosts is detrimental to parasite multiplication and Chagasic cardiomyopathy disease progression. This provides a novel framework for the understanding of Chagas disease pathogenesis.

The Intracellular Amastigote of Trypanosoma cruzi Maintains an Actively Beating Flagellum.

Throughout its complex life cycle, the uniflagellate parasitic protist, Trypanosoma cruzi, adapts to different host environments by transitioning between elongated motile extracellular stages and a nonmotile intracellular amastigote stage that replicates in the cytoplasm of mammalian host cells. Intracellular T. cruzi amastigotes retain a short flagellum that extends beyond the opening of the flagellar pocket with access to the extracellular milieu. Contrary to the long-held view that the T. cruzi amastigote flagellum is inert, we report that this organelle is motile and displays quasiperiodic beating inside mammalian host cells. Kymograph analysis determined an average flagellar beat frequency of ~0.7 Hz for intracellular amastigotes and similar beat frequencies for extracellular amastigotes following their isolation from host cells. Inhibitor studies reveal that flagellar motility in T. cruzi amastigotes is critically dependent on parasite mitochondrial oxidative phosphorylation. These novel observations reveal that flagellar motility is an intrinsic property of T. cruzi amastigotes and suggest that this organelle may play an active role in the parasite infection process. IMPORTANCE Understanding the interplay between intracellular pathogens and their hosts is vital to the development of new treatments and preventive strategies. The intracellular "amastigote" stage of the Chagas disease parasite, Trypanosoma cruzi, is a critical but understudied parasitic life stage. Previous work established that cytosolically localized T. cruzi amastigotes engage physically and selectively with host mitochondria using their short, single flagellum. The current study was initiated to examine the dynamics of the parasite flagellum-host mitochondrial interaction through live confocal imaging and led to the unexpected discovery that the T. cruzi amastigote flagellum is motile.

Extremely divergent COI sequences within an amphipod species complex: A possible role for endosymbionts?

Some heritable endosymbionts can affect host mtDNA evolution in various ways. Amphipods host diverse endosymbionts, but whether their mtDNA has been influenced by these endosymbionts has yet to be considered. Here, we investigated the role of endosymbionts (microsporidians and Rickettsia) in explaining highly divergent COI sequences in Paracalliope fluviatilis species complex, the most common freshwater amphipods in New Zealand. We first contrasted phylogeographic patterns using COI, ITS, and 28S sequences. While molecular species delimitation methods based on 28S sequences supported 3-4 potential species (N, C, SA, and SB) among freshwater lineages, COI sequences supported 17-27 putative species reflecting high inter-population divergence. The deep divergence between NC and S lineages (~20%; 28S) and the substitution saturation on the 3rd codon position of COI detected even within one lineage (SA) indicate a very high level of morphological stasis. Interestingly, individuals infected and uninfected by Rickettsia comprised divergent COI lineages in one of four populations tested, suggesting a potential influence of endosymbionts in mtDNA patterns. We propose several plausible explanations for divergent COI lineages, although they would need further testing with multiple lines of evidence. Lastly, due to common morphological stasis and the presence of endosymbionts, phylogeographic patterns of amphipods based on mtDNA should be interpreted with caution.

IFN-induced cell-autonomous immune mechanisms in the control of intracellular protozoa.

Vertebrate cells have evolved an elaborate multi-tiered intracellular surveillance system linked to downstream antimicrobial effectors to defend themselves from pathogens. This cellular self-defense system is referred to as cell-autonomous immunity. A wide array of cell-autonomous mechanisms operates to control intracellular pathogens including protozoa such as Toxoplasma gondii. Cell-autonomous immunity consists of antimicrobial defenses that are constitutively active in cells and those that are inducible typically in response to host cell activation. The IFN family of cytokines is an important stimulator of inducible cell-autonomous immunity. There are several hundred interferon-stimulated genes (ISGs); many of them have known roles in inducible cell-autonomous immune mechanisms. The importance of IFN-γ activation of cell-autonomous immunity is evidenced by the fact that many intracellular pathogens have evolved a diversity of molecular mechanisms to inhibit activation of infected cells through the JAK-STAT pathway in response to IFN-γ. The goal of this review is to provide a broad framework for understanding the elaborate system of cell-autonomous immunity that acts as a first line of defense between a host and intracellular parasites.

Differentiating Trypanosoma cruzi in a Host Mammalian Cell Imaged in Aqueous Liquid by Atmospheric Scanning Electron Microscopy.

Atmospheric Scanning Electron Microscopy (ASEM) is a powerful tool to observe a wet specimen at high resolution under atmospheric pressure. Here, we visualized a protozoan parasite Trypanosoma cruzi over the course of its infection cycle in the host mammalian cell. This is the first observation of intracellular parasite using a liquid-phase EM. Unlike regular SEM, aldehyde-fixed cell body of T. cruzi appears translucent, allowing the visualization of internal structures such as kinetoplast of trypomastigote and nucleus of amastigote. Plasma membrane of the host mammalian cell also appears translucent, which enabled direct observation of differentiating intracellular parasites and dynamic change of host cellular structures in their near-natural states. Various water-rich structures including micro- and macro- vesicles were visualized around T. cruzi. In addition, Correlative Light and Electron Microscopy exploiting open sample dish of ASEM allowed identification of parasite nucleus and transfected fluorescence-labeled parasites soon after internalization, while location of this morphological intermediate was otherwise obscure. Successful visualization of the differentiation of T. cruzi within the host cell demonstrated here opens up the possibility of using ASEM for observation of variety of intracellular parasites. IMPORTANCE Using Atmospheric Scanning Electron Microscopy (ASEM), we visualized interaction between infectious stage of Trypanosoma cruzi and completely intact host mammalian cell. Plasma membrane appears translucent under ASEM, which not only enables direct observation of T. cruzi within its host cell, but also reveals internal structures of the parasite itself. Sample deformation is minimal, since the specimen remains hydrated under atmospheric pressure at all times. This nature of ASEM, along with the open structure of ASEM sample dish, is suited for correlative light-electron microscopy, which can further be exploited in identification of fluorescent protein in the intracellular parasites.

Metabolic Plasticity Aids Amphotropism of Coxiella burnetii.

Coxiella burnetii, the causative agent of query (Q) fever in humans, is an obligate intracellular bacterium. C. burnetii can naturally infect a broad range of host organisms (e.g., mammals and arthropods) and cell types. This amphotropic nature of C. burnetii, in combination with its ability to utilize both glycolytic and gluconeogenic carbon sources, suggests that the pathogen relies on metabolic plasticity to replicate in nutritionally diverse intracellular environments. To test the significance of metabolic plasticity in C. burnetii host cell colonization, C. burnetii intracellular replication in seven distinct cell lines was compared between a metabolically competent parental strain and a mutant, CbΔpckA, unable to undergo gluconeogenesis. Both the parental strain and CbΔpckA mutant exhibited host cell-dependent infection phenotypes, which were influenced by alterations to host glycolytic or gluconeogenic substrate availability. Because the nutritional environment directly impacts host cell physiology, our analysis was extended to investigate the response of C. burnetii replication in mammalian host cells cultivated in a novel physiological medium based on the nutrient composition of mammalian interstitial fluid, interstitial fluid-modeled medium (IFmM). An infection model based on IFmM resulted in exacerbation of a replication defect exhibited by the CbΔpckA mutant in specific cell lines. The CbΔpckA mutant was also attenuated during infection of an animal host. Overall, the study underscores that gluconeogenic capacity aids C. burnetii amphotropism and that the amphotropic nature of C. burnetii should be considered when resolving virulence mechanisms in this pathogen.

Murine Irgm Paralogs Regulate Nonredundant Functions To Execute Host Defense to Toxoplasma gondii.

Gamma interferon (IFN-γ)-induced immunity-related GTPases (IRGs) confer cell-autonomous immunity to the intracellular protozoan pathogen Toxoplasma gondii. Effector IRGs are loaded onto the Toxoplasma-containing parasitophorous vacuole (PV), where they recruit ubiquitin ligases, ubiquitin-binding proteins, and IFN-γ-inducible guanylate-binding proteins (Gbps), prompting PV lysis and parasite destruction. Host cells lacking the regulatory IRGs Irgm1 and Irgm3 fail to load effector IRGs, ubiquitin, and Gbps onto the PV and are consequently defective for cell-autonomous immunity to Toxoplasma. However, the role of the third regulatory IRG, Irgm2, in cell-autonomous immunity to Toxoplasma has remained unexplored. Here, we report that Irgm2 unexpectedly plays a limited role in the targeting of effector IRGs, ubiquitin, and Gbps to the Toxoplasma PV. Instead, Irgm2 is instrumental in the decoration of PVs with γ-aminobutyric acid receptor-associated protein-like 2 (GabarapL2). Cells lacking Irgm2 are as defective for cell-autonomous host defense to Toxoplasma as pan-Irgm-/- cells lacking all three Irgm proteins, and Irgm2-/- mice succumb to Toxoplasma infections as readily as pan-Irgm-/- mice. These findings demonstrate that, relative to Irgm1 and Irgm3, Irgm2 plays a distinct but critically important role in host resistance to Toxoplasma.

Manipulation of Host Cell Organelles by Intracellular Pathogens.

Pathogenic intracellular bacteria, parasites and viruses have evolved sophisticated mechanisms to manipulate mammalian host cells to serve as niches for persistence and proliferation. The intracellular lifestyles of pathogens involve the manipulation of membrane-bound organellar compartments of host cells. In this review, we described how normal structural organization and cellular functions of endosomes, endoplasmic reticulum, Golgi apparatus, mitochondria, or lipid droplets are targeted by microbial virulence mechanisms. We focus on the specific interactions of Salmonella, Legionella pneumophila, Rickettsia rickettsii, Chlamydia spp. and Mycobacterium tuberculosis representing intracellular bacterial pathogens, and of Plasmodium spp. and Toxoplasma gondii representing intracellular parasites. The replication strategies of various viruses, i.e., Influenza A virus, Poliovirus, Brome mosaic virus, Epstein-Barr Virus, Hepatitis C virus, severe acute respiratory syndrome virus (SARS), Dengue virus, Zika virus, and others are presented with focus on the specific manipulation of the organelle compartments. We compare the specific features of intracellular lifestyle and replication cycles, and highlight the communalities in mechanisms of manipulation deployed.

Toxoplasma gondii Toxolysin 4 Contributes to Efficient Parasite Egress from Host Cells.

Egress from host cells is an essential step in the lytic cycle of T. gondii and other apicomplexan parasites; however, only a few parasite secretory proteins are known to affect this process. The putative metalloproteinase toxolysin 4 (TLN4) was previously shown to be an extensively processed microneme protein, but further characterization was impeded by the inability to genetically ablate TLN4. Here, we show that TLN4 has the structural properties of an M16 family metalloproteinase, that it possesses proteolytic activity on a model substrate, and that genetic disruption of TLN4 reduces the efficiency of egress from host cells. Complementation of the knockout strain with the TLN4 coding sequence significantly restored egress competency, affirming that the phenotype of the Δtln4 parasite was due to the absence of TLN4. This work identifies TLN4 as the first metalloproteinase and the second microneme protein to function in T. gondii egress. The study also lays a foundation for future mechanistic studies defining the precise role of TLN4 in parasite exit from host cells. IMPORTANCE After replicating within infected host cells, the single-celled parasite Toxoplasma gondii must rupture out of such cells in a process termed egress. Although it is known that T. gondii egress is an active event that involves disruption of host-derived membranes surrounding the parasite, very few proteins that are released by the parasite are known to facilitate egress. In this study, we identify a parasite secretory protease that is necessary for efficient and timely egress, laying the foundation for understanding precisely how this protease facilitates T. gondii exit from host cells.

Generation of a Microsporidia Species Attribute Database and Analysis of the Extensive Ecological and Phenotypic Diversity of Microsporidia.

Microsporidia are a large group of fungus-related obligate intracellular parasites. Though many microsporidia species have been identified over the past 160 years, depiction of the full diversity of this phylum is lacking. To systematically describe the characteristics of these parasites, we created a database of 1,440 species and their attributes, including the hosts they infect and spore characteristics. We find that microsporidia have been reported to infect 16 metazoan and 4 protozoan phyla, with smaller phyla being underrepresented. Most species are reported to infect only a single host, but those that are generalists are also more likely to infect a broader set of host tissues. Strikingly, polar tubes are threefold longer in species that infect tissues besides the intestine, suggesting that polar tube length is a determinant of tissue specificity. Phylogenetic analysis revealed four clades which each contain microsporidia that infect hosts from all major habitats. Although related species are more likely to infect similar hosts, we observe examples of changes in host specificity and convergent evolution. Taken together, our results show that microsporidia display vast diversity in their morphology and the hosts they infect, illustrating the flexibility of these parasites to evolve new traits. IMPORTANCE Microsporidia are a large group of parasites that cause death and disease in humans and many agriculturally important animal species. To fully understand the diverse properties of these parasites, we curated species reports from the last 160 years. Using these data, we describe when and where microsporidia were identified and what types of animals and host tissues these parasites infect. Microsporidia infect hosts using a conserved apparatus known as the polar tube. We observe that the length of this tube is correlated with the tissues that are being infected, suggesting that the polar tube controls where within the animals that the parasite infects. Finally, we show that microsporidia species often exist in multiple environments and are flexible in their ability to evolve new traits. Our study provides insight into the ecology and evolution of microsporidia and provides a useful resource to further understand these fascinating parasites.

Two parasites in one host: spatiotemporal dynamics and co-occurrence of Microsporidia and Rickettsia in an amphipod host.

Biological interactions can greatly influence the abundance of species. This is also true for parasitic species that share the same host. Microsporidia and Rickettsia are widespread intracellular parasites in populations of Paracalliope fluviatilis, the most common freshwater amphipods in New Zealand. Although both parasites coexist in many populations, it is unclear whether they interact with each other. Here, we investigated spatial−temporal dynamics and co-occurrence of the two parasites, Microsporidia and Rickettsia in P. fluviatilis hosts, across one annual cycle and in three different locations. Prevalence of both Microsporidia and Rickettsia changed over time. However, while the prevalence of Rickettsia varied significantly between sampling times, that of Microsporidia did not change significantly and remained relatively low. The two parasites therefore followed different temporal patterns. Also, the prevalence of both parasites differed among locations, though the two species reached their highest prevalence in different locations. Lastly, there was no evidence for positive or negative associations between the two parasite species; the presence of one parasite in an individual host does not appear to influence the probability of infection by the other parasite. Their respective prevalence may follow different patterns among populations on a larger spatial scale due to environmental heterogeneity across locations.

Revisiting the phylogeny of microsporidia.

Canonical microsporidians are a group of obligate intracellular parasites of a wide range of hosts comprising ~1,300 species of >220 genera. Microsporidians are related to fungi, and many characterised and uncharacterized groups closely related to them have been discovered recently, filling the knowledge gaps between them. These groups assigned to the superphylum Opisthosporidia have provided several important insights into the evolution of diverse intracellular parasitic lineages within the tree of eukaryotes. The most studied among opisthosporidians, canonical microsporidians, were known to science more than 160 years ago, however, the classification of canonical Microsporidia has been challenging due to common morphological homoplasy, and accelerated evolutionary rates. Instead of morphological characters, ssrRNA sequences have been used as the primary data for the classification of canonical microsporidians. Previous studies have produced a useful backbone of the microsporidian phylogeny, but provided only some nodal support, causing some confusion. Here, we reconstructed phylogenetic trees of canonical microsporidians using Bayesian and Maximum Likelihood inferences. We included rRNA sequences of 126 described/named genera, by far the broadest taxon coverage to date. Overall, our trees show similar topology and recovered four of the five main clades demonstrated in previous studies (Clades 1, 3, 4 and 5). Family level clades were well resolved within each major clade, but many were discordant with the recently revised classification. Therefore, revision and some reshuffling, especially within and between Clades 1 and 3 are required. We also reconstructed phylogenetic trees of Opisthosporidia to better integrate the evolutionary history of canonical microsporidians in a broader context. We discuss several traits shared only by canonical microsporidians that may have contributed to their striking ecological success in diverse metazoans. More targeted studies on the neglected host groups will be of value for a better understanding of the evolutionary history of these interesting intracellular parasites.

Molecular phylogeny and new light microscopic data of Metchnikovella spiralis (Microsporidia: Metchnikovellidae), a hyperparasite of eugregarine Polyrhabdina sp. from the polychaete Pygospio elegans.

Metchnikovellids are a deep-branching group of microsporidia, parasites of gregarines inhabiting the alimentary tract of polychaetes and some other invertebrates. The diversity and phylogeny of these hyperparasites remain poorly studied. Modern descriptions and molecular data are still lacking for many species. The results of a light microscopy study and molecular data for Metchnikovella spiralis Sokolova et al., 2014, a hyperparasite of the eugregarine Polyrhabdina sp., isolated from the polychaete Pygospio elegans, were obtained. The original description of M. spiralis was based primarily on the analysis of stained preparations and transmission electron microscopy images. Here, the species description was complemented with the results of in vivo observations and phylogenetic analysis based on the SSU rRNA gene. It was shown that in this species, free sporogony precedes sac-bound sporogony, as it occurs in the life cycle of most other metchnikovellids. Spore sacs are entwined with spirally wound cords, and possess only one polar plug. Phylogenetic analyses did not group M. spiralis with M. incurvata, another metchnikovellid from the same gregarine species, but placed it as a sister branch to Amphiacantha. The paraphyletic nature of the genus Metchnikovella was discussed. The taxonomic summary for M. spiralis was emended.

ISG15 Connects Autophagy and IFN-γ-Dependent Control of Toxoplasma gondii Infection in Human Cells.

The intracellular protozoan parasite Toxoplasma gondii is capable of infecting most nucleated cells, where it survives in a specially modified compartment called the parasitophorous vacuole (PV). Interferon gamma (IFN-γ) is the major cytokine involved in activating cell-autonomous immune responses to inhibit parasite growth within this intracellular niche. In HeLa cells, IFN-γ treatment leads to ubiquitination of susceptible parasite strains, recruitment of the adaptors p62 and NDP52, and engulfment in microtubule-associated protein 1 light chain 3 (LC3)-positive membranes that restrict parasite growth. IFN-γ-mediated growth restriction depends on core members of the autophagy (ATG) pathway but not the initiation or degradative steps in the process. To explore the connection between these different pathways, we used permissive biotin ligation to identify proteins that interact with ATG5 in an IFN-γ-dependent fashion. Network analysis of the ATG5 interactome identified interferon-stimulated gene 15 (ISG15), which is highly upregulated by IFN treatment, as a hub connecting the ATG complex with other IFN-γ-induced genes, suggesting that it forms a functional link between the pathways. Deletion of ISG15 resulted in impaired recruitment of p62, NDP52, and LC3 to the PV and loss of IFN-γ-restricted parasite growth. The function of ISG15 required conjugation, and a number of ISGylated targets overlapped with the IFN-γ-dependent ATG5 interactome, including the adapter p62. Collectively, our findings establish a role for ISG15 in connecting the ATG pathway with IFN-γ-dependent restriction of T. gondii in human cells.IMPORTANCE Interferon(s) provide the primary defense against intracellular pathogens, a property ascribed to their ability to upregulate interferon-stimulated genes. Due to the sequestered niche occupied by Toxoplasma gondii, the host has elaborated intricate ways to target the parasite within its vacuole. One such mechanism is the recognition by a noncanonical autophagy pathway that envelops the parasite-containing vacuole and stunts growth in human cells. Remarkably, autophagy-dependent growth restriction requires interferon-γ, yet none of the classical components of autophagy are induced by interferon. Our studies draw a connection between these pathways by demonstrating that the antiviral protein ISG15, which is normally upregulated by interferons, links the autophagy-mediated control to ubiquitination of the vacuole. These findings suggest a similar link between interferon-γ signaling and autophagy that may underlie defense against other intracellular pathogens.

Fundamental Difficulties Prevent the Reconstruction of the Deep Phylogeny of Viruses.

The extension of virology beyond its traditional medical, veterinary, or agricultural applications, now called environmental virology, has shown that viruses are both the most numerous and diverse biological entities on Earth. In particular, virus isolations from unicellular eukaryotic hosts (heterotrophic and photosynthetic protozoans) revealed numerous viral types previously unexpected in terms of virion structure, gene content, or mode of replication. Complemented by large-scale metagenomic analyses, these discoveries have rekindled interest in the enigma of the origin of viruses, for which a description encompassing all their diversity remains not available. Several laboratories have repeatedly tackled the deep reconstruction of the evolutionary history of viruses, using various methods of molecular phylogeny applied to the few shared "core" genes detected in certain virus groups (e.g., the Nucleocytoviricota). Beyond the practical difficulties of establishing reliable homology relationships from extremely divergent sequences, I present here conceptual arguments highlighting several fundamental limitations plaguing the reconstruction of the deep evolutionary history of viruses, and even more the identification of their unique or multiple origin(s). These arguments also underline the risk of establishing premature high level viral taxonomic classifications. Those limitations are direct consequences of the random mechanisms governing the reductive/retrogressive evolution of all obligate intracellular parasites.

Computational Modeling and Analysis to Predict Intracellular Parasite Epitope Characteristics Using Random Forest Technique.

BACKGROUND: In a new approach, computational methods are used to design and evaluate the vaccine. The aim of the current study was to develop a computational tool to predict epitope candidate vaccines to be tested in experimental models. METHODS: This study was conducted in the School of Allied Medical Sciences, and Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran in 2018. The random forest which is a classifier method was used to design computer-based tool to predict immunogenic peptides. Data was used to check the collected information from the IEDB, UniProt, and AAindex database. Overall, 1,264 collected data were used and divided into three parts; 70% of the data was used to train, 15% to validate and 15% to test the model. Five-fold cross-validation was used to find optimal hyper parameters of the model. Common performance metrics were used to evaluate the developed model. RESULTS: Twenty seven features were identified as more important using RF predictor model and were used to predict the class of peptides. The RF model improves the performance of predictor model in comparison with the other predictor models (AUC±SE: 0.925±0.029). Using the developed RF model helps to identify the most likely epitopes for further experimental studies. CONCLUSION: The current developed random forest model is able to more accurately predict the immunogenic peptides of intracellular parasites.

Mucosal microbial parasites/symbionts in health and disease: an integrative overview.

Microbial parasites adapted to thrive at mammalian mucosal surfaces have evolved multiple times from phylogenetically distant lineages into various extracellular and intracellular life styles. Their symbiotic relationships can range from commensalism to parasitism and more recently some host-parasites interactions are thought to have evolved into mutualistic associations too. It is increasingly appreciated that this diversity of symbiotic outcomes is the product of a complex network of parasites-microbiota-host interactions. Refinement and broader use of DNA based detection techniques are providing increasing evidence of how common some mucosal microbial parasites are and their host range, with some species being able to swap hosts, including from farm and pet animals to humans. A selection of examples will illustrate the zoonotic potential for a number of microbial parasites and how some species can be either disruptive or beneficial nodes in the complex networks of host-microbe interactions disrupting or maintaining mucosal homoeostasis. It will be argued that mucosal microbial parasitic diversity will represent an important resource to help us dissect through comparative studies the role of host-microbe interactions in both human health and disease.