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We are entering a Platinum Age of Virus Discovery, an era marked by exponential growth in the discovery of virus biodiversity, and driven by advances in metagenomics and computational analysis. In the ecosystem of a human (or any animal) there are more species of viruses than simply those directly infecting the animal cells. Viruses can infect all organisms constituting the microbiome, including bacteria, fungi, and unicellular parasites. Thus the complexity of possible interactions between host, microbe, and viruses is unfathomable. To understand this interaction network we must employ computationally assisted virology as a means of analyzing and interpreting the millions of available samples to make inferences about the ways in which viruses may intersect human health. From a computational viral screen of human neuronal datasets, we identified a novel narnavirus Apocryptovirus odysseus (Ao) which likely infects the neurotropic parasite Toxoplasma gondii. Previously, several parasitic protozoan viruses (PPVs) have been mechanistically established as triggers of host innate responses, and here we present in silico evidence that Ao is a plausible pro-inflammatory factor in human and mouse cells infected by T. gondii. T. gondii infects billions of people worldwide, yet the prognosis of toxoplasmosis disease is highly variable, and PPVs like Ao could function as a hitherto undescribed hypervirulence factor. In a broader screen of over 7.6 million samples, we explored phylogenetically proximal viruses to Ao and discovered nineteen Apocryptovirus species, all found in libraries annotated as vertebrate transcriptome or metatranscriptomes. While samples containing this genus of narnaviruses are derived from sheep, goat, bat, rabbit, chicken, and pigeon samples, the presence of virus is strongly predictive of parasitic Apicomplexa nucleic acid co-occurrence, supporting the fact that Apocryptovirus is a genus of parasite-infecting viruses. This is a computational proof-of-concept study in which we rapidly analyze millions of datasets from which we distilled a mechanistically, ecologically, and phylogenetically refined hypothesis. We predict that this highly diverged Ao RNA virus is biologically a T. gondii infection, and that Ao, and other viruses like it, will modulate this disease which afflicts billions worldwide.

Ciliophora are one of the most diverse phyla of unicellular eukaryotes. Members of this group are widely distributed and have been found in several environments including those that are anaerobic. However, the paucity of sufficient omics data for the major classes of the phylum Ciliophora, especially the anaerobic ciliate lineages, has been a serious impediment to research thus far. In this study, omics data were expanded to include representatives of 16 ciliate classes (excluding Cariacotrichea and Copemetopea) using single‐cell sequencing techniques. Using omics data of 57 ciliates and two apicomplexans, we conducted a comparative genomics study. Phylogenomic analysis based on 156 orthologous genes showed that: there are two superclades within the subphylum Intramacronucleata, i.e., Colpodea + Oligohymenophorea + Nassophorea + Plagiopylea + Prostomatea + Phyllopharyngea (CONThreeP), and Spirotrichea + Armophorea + Litostomatea + Odontostomatea + Muranotrichea + Parablepharismea (SALOMP); with the exception of Armophorea, all ciliate classes are monophyletic; neither Metopus nor Plagiopyla is monophyletic; Paraspathidium has a close affiliation with prostomateans. The results of the divergence time estimations indicated that: the radiation of Ciliophora occurred ca. 1067 million years ago (Mya); the SALOMP and CONThreeP lineages diverged from the intramacronucleatean common ancestor ca. 892 Mya and 851 Mya, respectively; the split between Muranotrichea and Parablepharismea occurred ca. 712 Mya; Odontostomatea are the youngest class, with a crown age estimated at ca. 19 Mya. In addition, we profiled stop codon usage and programmed ribosomal frameshifting (PRF) events. Our analyses revealed that: most of the species investigated use either TAA or TGA as the biased stop codon; stop codon usage in Paraspathidium sp. resembles that of Prostomatea; almost all ciliates included in the analyses have prevalent +1 and −1 PRF events. Finally, through GO enrichment analysis for group‐shared orthogroups of five anaerobic ciliate classes, we successfully identified the biological process and molecular function GO terms that were related to the adaptation of this particular group of ciliates to anaerobic environments. Together, these findings provide new insights into the evolutionary history of the Ciliophora and deepen our understanding of the adaptation of anaerobic ciliate groups to anaerobic environments from an evolutionary perspective. [ABSTRACT FROM AUTHOR]

Background: Coccidia are a group of intracellular protozoal parasites within the phylum Apicomplexa. Eimeria tenella, one of the species that cause intestinal coccidiosis in poultry, can cause significant mortality and morbidity. Diploid oocysts of Eimeria species are shed in the feces of an infected host and must sporulate to achieve infectivity. This process results in eight haploid infectious units, called sporozoites, held within a single oocyst. Each Eimeria spp. parasite possesses a single apicoplast and a single mitochondrion, both of which carry multiple copies of their respective organellar genomes. Reports of copy numbers of organellar genomes have varied widely. Methods: We report the application of quantitative polymerase chain reaction (qPCR), supported by next-generation sequencing, for the quantification of the extranuclear genomes relative to the nuclear genome over the course of sporulation and following its completion. Results: At 64 elapsed hours, 93.0% of oocysts were fully sporulated; no increase in percent sporulation was observed after this time. Apicoplast relative genome copy number showed several significant shifts up to 72 elapsed hours, after which no significant shifts were observed. Oocysts were shed with approximately 60% the amount of apicoplast DNA present at 72 h, after which point no significant shifts in apicoplast genome relative abundance occurred. Mitogenome relative copy number showed only two significant shifts, from 16 to 24 elapsed hours and from 24 to 32 elapsed hours. Oocysts were shed with approximately 28% the amount of mitochondrial DNA that was present at the time sporulation was deemed morphologically complete, at 64 elapsed hours. Conclusions: The characterization of the dynamics of genome abundance in exogenous stages sheds new light on the basic biology of Eimeria spp. and supports the use of extranuclear targets for molecular modes of parasite quantification and identification with improved sensitivity and accuracy. [ABSTRACT FROM AUTHOR]

Background: Human cryptosporidiosis is distributed worldwide, and it is recognised as a leading cause of acute diarrhoea and death in infants in low- and middle-income countries. Besides immune status, the higher incidence and severity of this gastrointestinal disease in young children could also be attributed to the digestive environment. For instance, human gastrointestinal physiology undergoes significant changes with age, however the role this variability plays in Cryptosporidium parvum pathogenesis is not known. In this study, we analysed for the first time the impact of digestive physicochemical parameters on C. parvum infection in a human and age-dependent context using a dynamic in vitro gastrointestinal model. Results: Our results showed that the parasite excystation, releasing sporozoites from oocysts, occurs in the duodenum compartment after one hour of digestion in both child (from 6 months to 2 years) and adult experimental conditions. In the child small intestine, slightly less sporozoites were released from excystation compared to adult, however they exhibited a higher luciferase activity, suggesting a better physiological state. Sporozoites collected from the child jejunum compartment also showed a higher ability to invade human intestinal epithelial cells compared to the adult condition. Global analysis of the parasite transcriptome through RNA-sequencing demonstrated a more pronounced modulation in ileal effluents compared to gastric ones, albeit showing less susceptibility to age-related digestive condition. Further analysis of gene expression and enriched pathways showed that oocysts are highly active in protein synthesis in the stomach compartment, whereas sporozoites released in the ileum showed downregulation of glycolysis as well as strong modulation of genes potentially related to gliding motility and secreted effectors. Conclusions: Digestion in a sophisticated in vitro gastrointestinal model revealed that invasive sporozoite stages are released in the small intestine, and are highly abundant and active in the ileum compartment, supporting reported C. parvum tissue tropism. Our comparative analysis suggests that physicochemical parameters encountered in the child digestive environment can influence the amount, physiological state and possibly invasiveness of sporozoites released in the small intestine, thus potentially contributing to the higher susceptibility of young individuals to cryptosporidiosis. [ABSTRACT FROM AUTHOR]

Apicomplexan parasites possess several specialized structures to invade their host cells and replicate successfully. One of these is the inner membrane complex (IMC), a peripheral membrane-cytoskeletal system underneath the plasma membrane. It is composed of a series of flattened, membrane-bound vesicles and a cytoskeletal subpellicular network (SPN) comprised of intermediate filament-like proteins called alveolins. While the alveolin proteins are conserved throughout the Apicomplexa and the broader Alveolata, their precise functions and interactions remain poorly understood. Here, we describe the function of one of these alveolin proteins in Toxoplasma, IMC6. Disruption of IMC6 resulted in striking morphological defects that led to aberrant invasion and replication but surprisingly minor effects on motility. Deletion analyses revealed that the alveolin domain alone is largely sufficient to restore localization and partially sufficient for function. As this highlights the importance of the IMC6 alveolin domain, we implemented unnatural amino acid photoreactive crosslinking to the alveolin domain and identified multiple binding interfaces between IMC6 and 2 other cytoskeletal IMC proteins—IMC3 and ILP1. This provides direct evidence of protein–protein interactions in the alveolin domain and supports the long-held hypothesis that the alveolin domain is responsible for filament formation. Collectively, our study features the conserved alveolin proteins as critical components that maintain the parasite's structural integrity and highlights the alveolin domain as a key mediator of SPN architecture. Picomplexan parasites require specialized structures like the inner membrane complex to invade host cells and replicate. This study shows that the alveolin protein IMC6 is crucial for maintaining cellular morphology, invasion and successful replication in Toxoplasma gondii. [ABSTRACT FROM AUTHOR]

Cytokinetic abscission marks the final stage of cell division, during which the daughter cells physically separate through the generation of new barriers, such as the plasma membrane or cell wall. While the contractile ring plays a central role during cytokinesis in bacteria, fungi and animal cells, the process diverges in Apicomplexa. In Toxoplasma gondii, two daughter cells are formed within the mother cell by endodyogeny. The mechanism by which the progeny cells acquire their plasma membrane during the disassembly of the mother cell, allowing daughter cells to emerge, remains unknown. Here we identify and characterize five T. gondii proteins, including three protein phosphatase 2A subunits, which exhibit a distinct and dynamic localization pattern during parasite division. Individual downregulation of these proteins prevents the accumulation of plasma membrane at the division plane, preventing the completion of cellular abscission. Remarkably, the absence of cytokinetic abscission does not hinder the completion of subsequent division cycles. The resulting progeny are able to egress from the infected cells but fail to glide and invade, except in cases of conjoined twin parasites. Synopsis: Cytokinetic abscission in Toxoplasma gondii is dependent on the accumulation of membrane material at the division plane. Instead of the ESCRT-III machinery, this parasite and other apicomplexans have evolved a specific set of Daughter Cell Scaffold proteins that include PP2A-2 subunits and are specifically required for this process. The dynamic localization of DCS and PP2A-2 suggests that they act early during division, while affecting the daughter cell separation at the end of the endodyogeny. Expression of all DCS proteins and PP2A-2 subunits is required for proper cellular abscission. Depletion of DCS or PP2A-2 proteins leads to the formation of a parasite syncytium that is able to egress but shows defects in motility and invasion. Syncytia consisting of two conjoined parasites can invade the host cell and establish a new lytic cycle. A set of five Toxoplasma gondii proteins is crucial for the buildup of the plasma membrane at the division plane, ensuring the successful completion of cellular abscission. [ABSTRACT FROM AUTHOR]

Microeukaryotes are a diverse and often overlooked group of microbes that are important in food webs and other ecological linkages. Little is known about microeukaryotes associated with aquatic invertebrates, although filter feeders such as mussels are likely to take in and potentially retain microeukaryotes in their gut while feeding. Microeukaryotes such as apicomplexans have been reported in marine mussel species, but no studies have examined the presence of these microorganisms in freshwater mussels or how they relate to mussel host species or environmental conditions. In this study, microbial community DNA was extracted from the gut tissue of over 300 freshwater mussels, representing 22 species collected from rivers in the southeastern USA. Microeukaryote DNA was detected using PCR amplification, followed by the sequencing of positive amplicons. Microeukaryotes were found in 167 individual mussels (53%) of those tested. Amplicons included dinoflagellates/algae that differed between mussel species and are likely food sources that were distinct from those found in water and sediment samples analyzed concurrently. A total of 5% of the positive amplicons were non-photosynthetic alveolates that could represent parasitic microeukaryotes. Understanding the distribution of microeukaryotes in the freshwater mussel gut microbiome could further our understanding of the ongoing decline of mussel populations. [ABSTRACT FROM AUTHOR]

Purpose: Nothing is known about coccidians (Apicomplexa: Eimeriidae) from the Pacific blue-tailed skink, Emoia caeruleocauda. Here, we report mensural and morphometric data on a new species of Isospora from E. caeruleocauda from Guam, US Territory. Methods: Feces from four E. caeruleocauda collected by hand in November 2023 were placed in individual vials containing 2.5% potassium dichromate. They were examined for sporulated oocysts after flotation in Sheather's sugar solution, measured, and photographed. Results: A single (25%) E. caeruleocauda was found to be passing oocysts representing a new species of Isospora. Oocysts of Isospora guamensis n. sp. are ellipsoidal to ovoidal with a bi-layered wall, measure (L × W) 16.5 × 11.8 μm, and have a length/width (L/W) ratio of 1.4; a micropyle and an oocyst residuum were absent but a polar granule was present. Sporocysts are ovoidal and measure 9.4 × 6.5 μm, L/W 1.4; Stieda and sub-Stieda bodies were present but a para-Stieda body was absent. The sporocyst residuum is composed various-sized granules in a compact rounded or irregular mass, sometimes dispersed between the sporozoites. The new species can be differentiated from all other isosporans from skinks by possessing the smallest oocysts known from this host family. Conclusion: This is the first time an isosporan coccidian has been reported from E. caeruleocauda as well as the first report of a coccidian from a Guam-inhabiting skink. [ABSTRACT FROM AUTHOR]

Abstract Background Coccidia are a group of intracellular protozoal parasites within the phylum Apicomplexa. Eimeria tenella, one of the species that cause intestinal coccidiosis in poultry, can cause significant mortality and morbidity. Diploid oocysts of Eimeria species are shed in the feces of an infected host and must sporulate to achieve infectivity. This process results in eight haploid infectious units, called sporozoites, held within a single oocyst. Each Eimeria spp. parasite possesses a single apicoplast and a single mitochondrion, both of which carry multiple copies of their respective organellar genomes. Reports of copy numbers of organellar genomes have varied widely. Methods We report the application of quantitative polymerase chain reaction (qPCR), supported by next-generation sequencing, for the quantification of the extranuclear genomes relative to the nuclear genome over the course of sporulation and following its completion. Results At 64 elapsed hours, 93.0% of oocysts were fully sporulated; no increase in percent sporulation was observed after this time. Apicoplast relative genome copy number showed several significant shifts up to 72 elapsed hours, after which no significant shifts were observed. Oocysts were shed with approximately 60% the amount of apicoplast DNA present at 72 h, after which point no significant shifts in apicoplast genome relative abundance occurred. Mitogenome relative copy number showed only two significant shifts, from 16 to 24 elapsed hours and from 24 to 32 elapsed hours. Oocysts were shed with approximately 28% the amount of mitochondrial DNA that was present at the time sporulation was deemed morphologically complete, at 64 elapsed hours. Conclusions The characterization of the dynamics of genome abundance in exogenous stages sheds new light on the basic biology of Eimeria spp. and supports the use of extranuclear targets for molecular modes of parasite quantification and identification with improved sensitivity and accuracy. Graphical Abstract

Abstract Background Human cryptosporidiosis is distributed worldwide, and it is recognised as a leading cause of acute diarrhoea and death in infants in low- and middle-income countries. Besides immune status, the higher incidence and severity of this gastrointestinal disease in young children could also be attributed to the digestive environment. For instance, human gastrointestinal physiology undergoes significant changes with age, however the role this variability plays in Cryptosporidium parvum pathogenesis is not known. In this study, we analysed for the first time the impact of digestive physicochemical parameters on C. parvum infection in a human and age-dependent context using a dynamic in vitro gastrointestinal model. Results Our results showed that the parasite excystation, releasing sporozoites from oocysts, occurs in the duodenum compartment after one hour of digestion in both child (from 6 months to 2 years) and adult experimental conditions. In the child small intestine, slightly less sporozoites were released from excystation compared to adult, however they exhibited a higher luciferase activity, suggesting a better physiological state. Sporozoites collected from the child jejunum compartment also showed a higher ability to invade human intestinal epithelial cells compared to the adult condition. Global analysis of the parasite transcriptome through RNA-sequencing demonstrated a more pronounced modulation in ileal effluents compared to gastric ones, albeit showing less susceptibility to age-related digestive condition. Further analysis of gene expression and enriched pathways showed that oocysts are highly active in protein synthesis in the stomach compartment, whereas sporozoites released in the ileum showed downregulation of glycolysis as well as strong modulation of genes potentially related to gliding motility and secreted effectors. Conclusions Digestion in a sophisticated in vitro gastrointestinal model revealed that invasive sporozoite stages are released in the small intestine, and are highly abundant and active in the ileum compartment, supporting reported C. parvum tissue tropism. Our comparative analysis suggests that physicochemical parameters encountered in the child digestive environment can influence the amount, physiological state and possibly invasiveness of sporozoites released in the small intestine, thus potentially contributing to the higher susceptibility of young individuals to cryptosporidiosis.

ImportanceThe classical depiction of the Toxoplasma lifecycle is bradyzoite excystation conversion to tachyzoites, cell lysis, and immune control, followed by the reestablishment of bradyzoites and cysts. In contrast, we show that tachyzoite growth slows independent of the host immune response at a predictable time point following excystation. Furthermore, we demonstrate a host cell-dependent pathway of continuous amplification of the cyst-forming bradyzoite population. The developmental plasticity of the excysted bradyzoites further underlines the critical role the cyst plays in the flexibility of the lifecycle of this ubiquitous parasite. This revised model of Toxoplasma recrudescence uncovers previously unknown complexity in the clinically important bradyzoite stage of the parasite, which opens the door to further study these novel developmental features of the Toxoplasma intermediate life cycle.

Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and Escherichia coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5 Å resolution revealing the inhibitor binding mode. In addition, we biologically characterize reverse α-phenyl-β-thia and β-oxa fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast with fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio) methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent antitoxoplasma DXR inhibitors. [ABSTRACT FROM AUTHOR]

Simple Summary: Sarcocystis parasites (Apicomplexa: Sarcocystidae) exhibit a complex life cycle necessitating two hosts and infect a wide range of mammals, birds, and reptiles. Birds of prey, positioned at the apex of the food chain, serve as definitive hosts for various Sarcocystis species. This study focuses on the molecular identification of Sarcocystis species in the intestines of the Common Buzzard (Buteo buteo). For this purpose, the small intestines of 30 birds were tested. Microscopic examination of intestinal scrapings and molecular analysis showed that 73.3% of the birds examined were infected with Sarcocystis spp. Nine Sarcocystis species using birds or rodents as their intermediate hosts were confirmed in the small intestines of Common Buzzards by means of DNA analysis. The highest detection rates were established for S. glareoli (53.3%) forming cysts in the brains of rodents and for S. halieti (36.7%) forming sarcocysts in the muscles and brains of birds. In conclusion, the Common Buzzard can transmit numerous Sarcocystis spp., including pathogenic ones. The common Buzzard (Buteo buteo) was previously shown to transmit two Sarcocystis species (S. glareoli and S. microti) forming cysts in the brains of rodents. Due to a lack of research, the richness of Sarcocystis species spread by these birds of prey is expected to be much higher. A total of 30 samples of the small intestine of the Common Buzzard were collected in Lithuania and subjected to Sarcocystis species identification based on nested PCR of 28S rRNA and ITS1, following the sequencing of amplified DNA fragments. Six known Sarcocystis spp., S. cornixi, S. glareoli, S. halieti, S. kutkienae, S. turdusi, and S. wobeseri, along with three genetically distinct species (Sarcocystis sp. Rod3, Sarcocystis sp. Rod4, and Sarcocystis sp. Rod5), were identified. Phylogenetically, these three potentially new species clustered with Sarcocystis spp. characterised by a rodents-birds life cycle. Sarcocystis spp. employing rodents and birds as their intermediate hosts were detected in 66.7% and 50.0% of samples, respectively. These findings are consistent with the diet preferences of Common Buzzards. Notably, co-infections with two or more species were observed in a half of the samples. Altogether, the obtained results indicate that the Common Buzzard could serve as definitive host of various Sarcocystis species. [ABSTRACT FROM AUTHOR]

Apicomplexan parasites balance proliferation, persistence, and spread in their metazoan hosts. AGC kinases, such as PKG, PKA, and the PDK1 ortholog SPARK, integrate environmental signals to toggle parasites between replicative and motile life stages. Recent studies have cataloged pathways downstream of apicomplexan PKG and PKA; however, less is known about the global integration of AGC kinase signaling cascades. Here, conditional genetics coupled to unbiased proteomics demonstrates that SPARK complexes with an elongin-like protein to regulate the stability of PKA and PKG in the model apicomplexan Toxoplasma gondii. Defects attributed to SPARK depletion develop after PKG and PKA are down-regulated. Parasites lacking SPARK differentiate into the chronic form of infection, which may arise from reduced activity of a coccidian-specific PKA ortholog. This work delineates the signaling topology of AGC kinases that together control transitions within the asexual cycle of this important family of parasites. [ABSTRACT FROM AUTHOR]

Rhoptries are specialized secretory organelles conserved across the Apicomplexa phylum, essential for host cell invasion and critical for subverting of host cellular and immune functions. They contain proteins and membranous materials injected directly into the host cells, participating in parasitophorous vacuole formation. Toxoplasma gondii tachyzoites harbor 8 to 12 rhoptries, 2 of which are docked to an apical vesicle (AV), a central element associated with a rhoptry secretory apparatus prior to injection into the host cell. This parasite is also equipped with 5 to 6 microtubule-associated vesicles, presumably serving as AV replenishment for iterative rhoptry discharge. Here, we characterized a rhoptry protein, rhoptry discharge factor 3 (RDF3), crucial for rhoptry discharge and invasion. RDF3 enters the secretory pathway, localizing near the AV and associated with the rhoptry bulb. Upon invasion, RDF3 dynamically delocalizes, suggesting a critical role at the time of rhoptry discharge. Cryo-electron tomography analysis of RDF3-depleted parasites reveals irregularity in microtubule-associated vesicles morphology, presumably impacting on their preparedness to function as an AV. Our findings suggest that RDF3 is priming the microtubule-associated vesicles for rhoptry discharge by a mechanism distinct from the rhoptry secretory apparatus contribution. Regulated secretion of rhoptry factors is essential for host cell invasion by Apicomplexan parasites. This study identifies the protein rhoptry discharge factor 3 (RDF3) of Toxoplasma gondii as crucial for invasion, by priming microtubule-associated vesicles for rhoptry discharge. [ABSTRACT FROM AUTHOR]

Simple Summary: Parasites can be transmitted from animals to humans by consumption of undercooked meat. One of these parasites is Toxoplasma gondii, a unicellular parasite that could cause severe disease in immunocompromised patients and foetal problems during pregnancy. T. gondii can be found in wild boar meat and be a source of infection for humans, particularly hunters and their inner circle of acquaintances. We tested serum samples of 42 free-ranging wild boars from Thuringia in central Germany during the hunting season of 2017/2018 and were able to detect antibodies against T. gondii in 18 of these animals (37.50%). The explicit prevalence of T. gondii specific antibodies points towards a risk of toxoplasmosis for people consuming game meat, particularly when it is not properly cooked. The hunting community as well as authorities should be aware of such possible exposures and infections. Additional studies among free-ranging wild boars would help to obtain more prevalence data from other areas of Germany and to better estimate the actual risk of T. gondii infection among the hunting community and game meat consumers' interface. Game meat is an important source of meat borne parasitic infections. Due to its omnivorous diet, the wild boar is an important host of zoonotic parasites such as Toxoplasma gondii. T. gondii can cause severe to fatal disease in immunosuppressed patients, as well as congenital disorders in foetus and neonates. Consumption of undercooked infected meat is a main source of T. gondii infection. Information about the risk of toxoplasmosis through game meat is scarce. We collected serum samples from 42 wild boars from the federal state of Thuringia (Germany) between December 2017 and February 2018. Identification of anti-T. gondii IgG antibodies was conducted using a commercial indirect ELISA kit. Seropositivity was confirmed in 18 of the 42 samples (37.50%). From these, the highest seroprevalence was found in adult animals. This study joins another single database from wild boars in Brandenburg. The necessity of a country-wide database regarding T. gondii prevalence in wild boar and other game meat is pivotal for a profound risk analysis with its consequential impact in future mean hygiene policies. [ABSTRACT FROM AUTHOR]

Neosporosis and toxoplasmosis are important parasitic causes of abortions in small ruminants. This study verified the occurrence of these diseases in sheep fetuses from Santa Catarina State, Southern Brazil from 2015 to 2022. Sheep fetuses were necropsied with organ sampling for histopathology, and polymerase chain reaction (PCR) for Neospora caninum and Toxoplasma gondii using the Nc5 and SAG2 targets, respectively, in frozen brain tissue. Microbiological culture and RT-PCR for Pestivirus were conducted to discard other abortion causes. One positive fetus for toxoplasmosis was genotyped using multiplex multilocus nested PCR-RFLP (Mn-PCR-RFLP) with ten genetic markers. Fifty-five sheep fetuses were evaluated, with 10 (18.2%) cases of neosporosis and 7 (12.7%) cases of toxoplasmosis, comprising six and four flocks, respectively. Macroscopically, neosporosis abortions exhibited fetal mummification, maceration, and arthrogryposis. Toxoplasmosis abortions showed fetal mummification and maceration. The neosporosis abortions included lymphoplasmacytic myositis (70%; 7/10) and myocarditis (60%; 6/10), in addition to necrotizing encephalitis and gliosis (50%; 5/10). Toxoplasmosis abortions included lymphoplasmacytic necrotizing encephalitis (71.4%; 5/7), lymphoplasmacytic myositis (42.8%, 3/7), and myocarditis (14.3%; 1/7). Through PCR, N. caninum and T. gondii were detected in 6 (60%) and 5 (71.4%) fetuses, respectively. In one fetus, T. gondii genotyping was conducted, which was characterized as atypical genotype ToxoDB #98. All of the cases were negative for Pestivirus and bacterial agents. This study establishes the occurrence of these diseases as causes of abortions, malformations, mummification, and fetal maceration in sheep, with the characterization of an atypical T. gondii genotype in one of the fetuses. [ABSTRACT FROM AUTHOR]

Eukaryotic translation initiation factors (eIFs) are crucial for initiating protein translation and ensuring the correct assembly of mRNA-ribosomal subunit complexes. In this study, we investigated the effects of deleting six eIFs in the apicomplexan parasite Toxoplasma gondii using the CRISPR-Cas9 system. We determined the subcellular localization of these eIFs using C-terminal endogenous tagging and immunofluorescence analysis. Four eIFs (RH::315150-6HA, RH::286090-6HA, RH::249370-6HA, and RH::211410-6HA) were localized in the cytoplasm, while RH::224235-6HA was localized in the apicoplast. Additionally, RH::272640-6HA was found in both the basal complex and the cytoplasm of T. gondii. Functional characterization of the six RHΔeIFs strains was conducted using plaque assay, cell invasion assay, intracellular growth assay and egress assay in vitro, and virulence assay in mice. Disruption of five eIF genes (RHΔ315150, RHΔ272640, RHΔ249370, RHΔ211410, and RHΔ224235) did not affect the ability of the T. gondii RH strain to invade, replicate, form plaques and egress in vitro, or virulence in Kunming mice (p > 0.05). However, the RHΔ286090 strain showed slightly reduced invasion efficiency and virulence (p < 0.01) compared to the other five RHΔeIFs strains and the wild-type strain. The disruption of the TGGT1_286090 gene significantly impaired the ability of tachyzoites to differentiate into bradyzoites in both type I RH and type II Pru strains. These findings reveal that the eukaryotic translation initiation factor TGGT1_286090 is crucial for T. gondii bradyzoite differentiation and may serve as a potential target for drug development and an attenuated vaccine against T. gondii. [ABSTRACT FROM AUTHOR]

Coccidiosis is one of the most prevalent diseases found in local rabbits (Oryctolagus cuniculus), which is caused by the Eimeria. The study aimed to more reliably identify Eimeria species (Eimeria magna) infecting Local Rabbits in Alkarg City, Saudi Arabia, based the method on the molecular properties and morphological and molecular biological techniques. Sub‐spheroidal oocysts measuring 21–27 × 12–16 (24 × 14.4) μm (20 n) and with a length/width (L/W) ratio of 0.9–1.1 (1.0) were identified by microscopic analysis of a fecal sample. Oocysts feature a bi‐layered wall that is 1.0–1.2 (1.1) μm thick. About two‐thirds of the wall's thickness is made up of a smooth outer layer. A polar granule is present, but neither a micropyle nor an oocyst residuum is present. The ovoidal sporozoites measure 15–18 × 8–11 (16.5 × 9.5) μm, have an L/W ratio of 1.6–1.8 (1.7), and take up around 21% of the oocyst's total surface. The mean size of the sub‐Stieda body is 1.4 × 2.3 μm, while the average size of the Stieda body is 0.9 × 1.8 μm. The para‐Stieda body is lacking. Sporocyst residuum appears membrane‐bound and has an uneven form made up of several granules. With two refractile bodies below the striations and pronounced striations at the more pointed end, sporozoites are vermiform, measuring an average of 11.6 × 4.0 μm. The results of the sequencing for the 18S rDNA gene confirmed the species of Eimeria parasites found in the host (rabbits). The current parasite species is closely related to the previously described and deposited E. magna and deeply embedded in the genus Eimeria (family Eimeriidae). According to the findings, single oocyst molecular identification of Eimeria may be accomplished through consistent use of the morphological and molecular results. It is possible to draw the conclusion that the current research supplies relevant facts that help assess the potential infection and future control measures against rabbit coccidiosis to reduce the financial losses that can be incurred by the rabbit industry in Saudi Arabia. [ABSTRACT FROM AUTHOR]

The peculiarities of variability of forms and sizes of the gregarine Stylocephalus oblongatus (Hammerschmidt, 1838) Watson, 1916 in the intestines of the Opatrum sabulosum (Linnaeus, 1761) from the sites of reclamation of lands disturbed by mining industry in the south of the area of the Steppe Dnipro were investigated. This is a new record for the protozoan fauna of Ukraine. The prevalence of host infection was 7.44 %. To determine the limits of variability, a sample of 71 specimens was used. Gamonts of S. oblongatus were analysed by 15 linear characteristics and 18 indices of the ratio of these characteristics. For linear parameters, the average coefficient of variation was 33 %. The minimum values of the coefficient of variation were characteristic for the lengths of the gamont, protomerite and their ratio. The principal component analysis identified 4 principal components, the value of which was greater than 1. Together they explained 73 % of the variability. Cluster analysis identified 4 clusters that can be explained as 2 morphotypes and 2 morphosubtypes. The greatest contribution to the overall variability of S. oblongatus gamonts is made by the position of the cell nucleus of the gamont in relation to the septum, the width of the septum and protomerite and the distance from the posterior end of the deuteromerite to the axis of its maximum width. The observed morphological differences can be explained by different degrees of host infection. It is important to compare the obtained information on intraspecific variability of S. oblongatus with similar data from natural ecosystems, as well as from agrocenoses and urban populations of O. sabulosum. [ABSTRACT FROM AUTHOR]

Colpodella species are free-living protists phylogenetically related to apicomplexans. Colpodella sp. have been detected in human and animal tissues, as well as in ticks and biting flies. The trophozoite and cyst stages of Colpodella species can be distinguished from stages of the prey Parabodo caudatus using Sam-Yellowe's trichrome staining. Colpodella species obtain nutrients by attaching to their prey, aspirating the prey's cytoplasmic contents into a posterior food vacuole and encysting. It is unclear whether both trophozoite and cyst stages are present in human and animal tissues. Molecular techniques have detected Colpodella species in human blood, cerebrospinal fluid, and in ticks and flies. However, no morphological information was reported to aid life-cycle stage identification of Colpodella species. This review discusses the increased reports of Colpodella species detection in animals and in arthropods and the need to identify stages present in human and animal tissues. We previously used Sam-Yellowe's trichrome staining to identify life-cycle stages of Colpodella sp. In this review, we examine the reports of Colpodella species detection in human and animal tissues to determine whether the identification of Colpodella species represents true infections or contaminations of samples collected during routine surveillance of piroplasm infections in animals and arthropods. This review also aims to provide insights regarding Colpodella, nutrient uptake, and the survival of Colpodella sp. within humans, animals, and arthropods, as well as whether the attachment of trophozoites to cells occurs in tissues leading to myzocytosis and endocytosis. [ABSTRACT FROM AUTHOR]

The apicomplexan intracellular parasite Toxoplasma gondii is a major food borne pathogen that is highly prevalent in the global population. The majority of the T. gondii proteome remains uncharacterized and the organization of proteins into complexes is unclear. To overcome this knowledge gap, we used a biochemical fractionation strategy to predict interactions by correlation profiling. To overcome the deficit of high-quality training data in non-model organisms, we complemented a supervised machine learning strategy, with an unsupervised approach, based on similarity network fusion. The resulting combined high confidence network, ToxoNet, comprises 2,063 interactions connecting 652 proteins. Clustering identifies 93 protein complexes. We identified clusters enriched in mitochondrial machinery that include previously uncharacterized proteins that likely represent novel adaptations to oxidative phosphorylation. Furthermore, complexes enriched in proteins localized to secretory organelles and the inner membrane complex, predict additional novel components representing novel targets for detailed functional characterization. We present ToxoNet as a publicly available resource with the expectation that it will help drive future hypotheses within the research community. Author summary: Toxoplasma gondii is a food-borne parasite that chronically infects 1 in 3 worldwide. It can cause blindness in children and is life-threatening during pregnancy and to those with compromised immune systems. During infection, Toxoplasma relies on the coordination of suites of proteins that allow it to invade host cells and persist. In this study we combined a sophisticated biochemical separation platform with a novel machine learning strategy to build a global roadmap that describes how parasite proteins work together as molecular machines to help drive infection. Our roadmap, which we term ToxoNet, details 2,063 connections between 652 proteins. These include proteins representing parasite-specific innovations that help Toxoplasma perform essential functions such as the generation of energy. Further, in addition to capturing proteins already implicated in host cell invasion pathways, we identified many additional components of these pathways that represent novel targets for future drug development strategies. [ABSTRACT FROM AUTHOR]

Introduction: Eimeria spp. are intracellular protozoan parasites of the phylum Apicomplexa causing economic losses to various wild and domestic animals. An eimerian species infecting Columba livia domestica was identified in this study. Methods: A total of 15 faecal samples were examined by floatation technique, a prevalence rate of 60% was reported. Eimerian oocysts were sporulated in 2.5% potassium dichromate solution then identified using morphological and molecular (DNA amplification of the 18S rRNA and ITS-1 genes) diagnostic techniques. Results: Sporulated oocysts were identified as Eimeria labbeana-like, after morphometry with typical bi-layered wall with spherical to subspherical oocysts morphology. A polar granule is present, but no micropyle or oocyst residuum. Sporocysts are elongated ovoidal with stieda body. Sporocyst residuum with many granules and sporozoites with refractile bodies and nucleus. Both 18S rRNA and ITS-1 sequences have been deposited in GenBank database. DNA sequences from the partial 18S rRNA generated from the oocysts were found to be related to eimerian and isosporan parasites found in domestic pigeons. For the first time, ITS-1 sequences for E. labbeana-like were provided. Conclusion: The necessity of using molecular techniques to describe pigeon intestinal coccidian parasites in conjunction with traditional morphology-based tools was emphasized in this work in order to understand the biology of such parasites. [ABSTRACT FROM AUTHOR]

Endosymbiotic organelles, such as mitochondria and plastids, contain own remnant genomes (nucleoids), whose variable abundance in cells may be adaptive to the physiological necessities and functions of the cells. Unicellular apicomplexan parasites contain one mitochondrium and one apicoplast with variable genome copy numbers. We measured the abundance of mitochondrial, apicoplast and nuclear genome copies in a set of avian blood samples infected with haemosporidian blood parasites, belonging to the three main genera Plasmodium, Haemoproteus and Leucocytozoon. We designed general primers suitable for qPCR, amplifying fragments of the mitogenome, plastome and nuclear genomes of avian malaria and related haemosporidian parasites. We measured the amplification of these fragments in 153 samples of 23 avian host species and infected with 33 parasitic lineages. We estimate on average several hundred mitochondrial genome copies and several tens of apicoplast copies per haploid gametocyte cell with substantial variation among samples. Host species appeared to differ in their mitogenome abundance while parasitic lineages differed in plastome abundance per cell (per nuclear copy signal). We did not find consistent differences between parasite genera or higher avian taxa. Parasite lineages and host bird species did not differ consistently in infection intensity, estimated from parasite to host nuclear signals, which may indicate that samples were taken at different stages of infection. However, this and similar results remain to be cross-validated with in-situ imaging techniques. The novel molecular tools introduced here offer avenues for the characterization of nucleoid abundance of haemosporidian parasites over environmental conditions and parasitic developmental stages. Such measures will improve our understanding of parasite physiology, ecology, the coadaptation and coevolution with hosts and suggest possible augmentations to standard methods in the research field. [ABSTRACT FROM AUTHOR]

Asexual replication of Plasmodium falciparum occurs via schizogony, wherein 16–36 daughter cells are produced within the parasite during one semi-synchronized cytokinetic event. Schizogony requires a divergent contractile ring structure known as the basal complex. Our lab has previously identified PfMyoJ (PF3D7_1229800) and PfSLACR (PF3D7_0214700) as basal complex proteins recruited midway through segmentation. Using ultrastructure expansion microscopy, we localized both proteins to a novel basal complex subcompartment. While both colocalize with the basal complex protein PfCINCH upon recruitment, they form a separate, more basal subcompartment termed the posterior cup during contraction. We also show that PfSLACR is recruited to the basal complex prior to PfMyoJ, and that both proteins are removed unevenly as segmentation concludes. Using live-cell microscopy, we show that actin dynamics are dispensable for basal complex formation, expansion, and contraction. We then show that EF-hand containing P. falciparum Centrin 2 partially localizes to this posterior cup of the basal complex and that it is essential for growth and replication, with variable defects in basal complex contraction and synchrony. Finally, we demonstrate that free intracellular calcium is necessary but not sufficient for basal complex contraction in P. falciparum. Thus, we demonstrate dynamic spatial compartmentalization of the Plasmodium falciparum basal complex, identify an additional basal complex protein, and begin to elucidate the unique mechanism of contraction utilized by P. falciparum, opening the door for further exploration of Apicomplexan cellular division. Author summary: Plasmodium falciparum causes the most dangerous type of malaria, a disease that significantly impacts global health. One of the challenges in attempting to develop new antimalarial treatments is a lack of knowledge about the basic biology of the parasite. Cell division is especially unique in Plasmodium, and the organization and mechanism of its cytokinetic ring, known as the basal complex, are poorly understood. Here, we use novel microscopy techniques to characterize the spatial and temporal complexity of the basal complex. We find that the basal complex is composed of two subcompartments which become distinct over the course of cell division, complicating the temporal map of the basal complex by observing slight differences in recruitment and removal time, and we localize a newly-identified, essential basal complex protein, PfCen2, to the basal subcompartment. Finally, we show that neither actin dynamics nor myosin activity are required for basal complex contraction, but that intracellular calcium signaling is. Our study thus provides new insights into the organization and mechanism of Plasmodium's cytokinetic ring and highlights a uniquely divergent, potentially exploitable aspect of Plasmodium cell biology. [ABSTRACT FROM AUTHOR]

A versatile division of apicomplexan parasites and a dearth of conserved regulators have hindered the progress of apicomplexan cell cycle studies. While most apicomplexans divide in a multinuclear fashion, Toxoplasma gondii tachyzoites divide in the traditional binary mode. We previously identified five Toxoplasma CDK-related kinases (Crk). Here, we investigated TgCrk4 and its cyclin partner TgCyc4. We demonstrated that TgCrk4 regulates conventional G2 phase processes, such as repression of chromosome rereplication and centrosome reduplication, and acts upstream of the spindle assembly checkpoint. The spatial TgCyc4 dynamics supported the TgCrk4–TgCyc4 complex role in the coordination of chromosome and centrosome cycles. We also identified a dominant TgCrk4–TgCyc4 complex interactor, TgiRD1 protein, related to DNA replication licensing factor CDT1 but played no role in licensing DNA replication in the G1 phase. Our results showed that TgiRD1 also plays a role in controlling chromosome and centrosome reduplication. Global phosphoproteome analyses identified TgCrk4 substrates, including TgORC4, TgCdc20, TgGCP2, and TgPP2ACA. Importantly, the phylogenetic and structural studies suggest the Crk4–Cyc4 complex is limited to a minor group of the binary dividing apicomplexans. Synopsis: Apicomplexan parasite division can yield between two and thousands of progenies, and how this process is regulated remains a mystery. This study identifies a presumed missing G2 phase, and the complex TgCrk4-TgCyc4-TgiRD1 that regulates G2 phase, in Toxoplasma gondii. Expression of the CDK-related kinase TgCrk4, of the cyclin TgCyc4, and of the inhibitor of the DNA and centrosome reduplication TgiRD1 is essential for binary division of T. gondii tachyzoites. Removing TgCrk4 and TgiRD1 triggers the reduplication of chromosomes and centrosomes, revealing their role as guardians of the "once per cycle" duplication rule. Although distantly related to fungal and metazoan CDT1, TgiRD1 does not license DNA replication in G1 phase. Comparative genomic studies imply that expression of the Crk4-Cyc4 complex is restricted to apicomplexan parasites undergoing binary division. A previously unrecognized G2 phase in apicomplexan parasites undergoing binary division depends on a novel CDK-related kinase, an atypical cyclin, and a Cdt1-related inhibitor of re-duplication. [ABSTRACT FROM AUTHOR]

A senile male black capuchin monkey (Sapajus nigritus) kept under human care in a Zoo was found dead after 2 weeks presenting signals of weight loss and hyporexia. Histopathological revealed a necrotizing encephalitis. Although it was not observed microscopically, Sarcocystis sp infection was detected in brain tissue from molecular assays. These infections have been rarely described in neotropical primates, particularly associated with tissue lesions. [ABSTRACT FROM AUTHOR]

Rabbit coccidiosis is caused by infection with one or, more commonly, several Eimeria species that parasitize the hepatobiliary ducts or intestinal epithelium of rabbits. Currently, there are eleven internationally recognized species of rabbit coccidia, with the complete mitochondrial (mt) genomes of six species commonly infecting rabbits having been sequenced and annotated. Eimeria kongi was initially discovered in 2011 and prompted a preliminary study on this species. Through traditional morphological analysis, E. kongi was identified as a novel species of rabbit coccidia. To further validate this classification, we sequenced and annotated its mitochondrial genome. The complete mt genome of E. kongi spans 6258 bp and comprises three cytochrome genes (cytb, cox1, cox3), fourteen gene fragments for the large subunit (LSU) rRNA, and nine gene fragments for the small subunit (SSU) rRNA, lacking transfer RNA (tRNA) genes. Moreover, phylogenetic analysis of the mitochondrial genome sequence of E. kongi revealed its clustering with six other species of rabbit coccidia into a monophyletic group. Additionally, E. irresidua and E. flavescens were grouped within the lineage lacking oocyst residuum, consistent with their morphological characteristics. Consistent with multiple molecular phylogenies, in this investigation, E. kongi was further confirmed as a new species of rabbit coccidia. Our research findings are of great significance for the classification of coccidia and for coccidiosis prevention and control in rabbits. [ABSTRACT FROM AUTHOR]

To analyze the presence of protozoan parasites in bird coprolites from the Tremembé Formation (Oligocene of the Taubaté Basin). Twenty avian coprolites embedded in pyrobituminous shale matrices. Samples were rehydrated and subjected to spontaneous sedimentation. Paleoparasitological analyses revealed oocysts compatible with the Eimeriidae family (Apicomplexa) and one single Archamoebae (Amoebozoa) cyst. The present work increases the amount of information about the spread of infections throughout the Cenozoic Era and reveals that the Brazilian paleoavifauna played an important role in the Apicomplexa and Amoebozoa life cycles. This is the first record of protozoans in avian coprolites from the Oligocene of Brazil. These findings can help in the interpretation of phylogenies of coccidian parasites of modern birds, as certain taxonomic characters observed in the Oligocene Protozoa characterize monophyletic groups in current molecular phylogenetic analyses. None of the oocysts were sporulated; therefore, it is not possible to identify the morphotypes to genus or species. Our results create new perspectives related to biogeographic studies of the parasitic groups described and may improve the understanding of the temporal amplitude of parasitic evolutionary relationships between Protozoans and birds. • Paleoparasitological analyzes in twenty Oligocene avian coprolites from Brazil were performed. • We report the first records of Eimeriidae and Archamoebae in avian coprolites from the Tremembé Formation. • The Oligocene birds from the Paraiba Valley were important for spreading of Eimeriidae and Archamoebae infections. [ABSTRACT FROM AUTHOR]

Apicomplexa comprise important pathogenic parasitic protists that heavily depend on lipid acquisition to survive within their human host cells. Lipid synthesis relies on the incorporation of an essential combination of fatty acids (FAs) either generated by a metabolically adaptable de novo synthesis in the parasite or by scavenging from the host cell. The incorporation of FAs into membrane lipids depends on their obligate metabolic activation by specific enzyme groups, acyl-CoA synthetases (ACSs). Each ACS has its own specificity, so it can fulfill specific metabolic functions. Whilst such functionalities have been well studied in other eukaryotic models, their roles and importance in Apicomplexa are currently very limited, especially for Toxoplasma gondii. Here, we report the identification of seven putative ACSs encoded by the genome of T. gondii (TgACS), which localize to different sub-cellular compartments of the parasite, suggesting exclusive functions. We show that the perinuclear/cytoplasmic TgACS3 regulates the replication and growth of Toxoplasma tachyzoites. Conditional disruption of TgACS3 shows that the enzyme is required for parasite propagation and survival, especially under high host nutrient content. Lipidomic analysis of parasites lacking TgACS3 reveals its role in the activation of host-derived FAs that are used for i) parasite membrane phospholipid and ii) storage triacylglycerol (TAG) syntheses, allowing proper membrane biogenesis of parasite progenies. Altogether, our results reveal the role of TgACS3 as the bulk FA activator for membrane biogenesis allowing intracellular division and survival in T. gondii tachyzoites, further pointing to the importance of ACS and FA metabolism for the parasite.

French Guiana, located in the Guiana Shield, is a natural reservoir for many zoonotic pathogens that are of considerable medical or veterinary importance. Until now, there has been limited data available on the description of parasites circulating in this area, especially on protozoan belonging to the phylum Apicomplexa; conversely, the neighbouring countries describe a high parasitic prevalence in animals and humans. Epidemiological surveillance is necessary, as new potentially virulent strains may emerge from these forest ecosystems, such as Amazonian toxoplasmosis. However, there is no standard tool for detecting protozoa in wildlife. In this study, we developed Meat-Borne-Parasite, a high-throughput meta-barcoding workflow for detecting Apicomplexa based on the Oxford Nanopore Technologies sequencing platform using the 18S gene of 14 Apicomplexa positive samples collected in French Guiana. Sequencing reads were then analysed with MetONTIIME pipeline. Thanks to a scoring rule, we were able to classify 10 samples out of 14 as Apicomplexa positive and reveal the presence of co-carriages. The same samples were also sequenced with the Illumina platform for validation purposes. For samples identified as Apicomplexa positive by both platforms, a strong positive correlation at up to the genus level was reported. Overall, the presented workflow represents a reliable method for Apicomplexa detection, which may pave the way for more comprehensive biomonitoring of zoonotic pathogens.

Abstract The use of fluorescent proteins (FPs) in Plasmodium parasites has been key to understand the biology of this obligate intracellular protozoon. FPs like the green fluorescent protein (GFP) enabled to explore protein localization, promoter activity as well as dynamic processes like protein export and endocytosis. Furthermore, FP biosensors have provided detailed information on physiological parameters at the subcellular level, and fluorescent reporter lines greatly extended the malariology toolbox. Still, in order to achieve optimal results, it is crucial to know exactly the properties of the FP of choice and the genetic scenario in which it will be used. This review highlights advantages and disadvantages of available landing sites and promoters that have been successfully applied for the ectopic expression of FPs in Plasmodium berghei and Plasmodium falciparum. Furthermore, the properties of newly developed FPs beyond DsRed and EGFP, in the visualization of cells and cellular structures as well as in the sensing of small molecules are discussed. Graphical Abstract

Toxoplasma gondii is a ubiquitous protozoan parasite that can reside long-term within hosts as intracellular tissue cysts comprised of chronic stage bradyzoites. To perturb chronic infection requires a better understanding of the cellular processes that mediate parasite persistence. Macroautophagy/autophagy is a catabolic and homeostatic pathway that is required for T. gondii chronic infection, although the molecular details of this process remain poorly understood. A key step in autophagy is the initial formation of the phagophore that sequesters cytoplasmic components and matures into a double-membraned autophagosome for delivery of the cargo to a cell’s digestive organelle for degradative recycling. While T. gondii appears to have a reduced repertoire of autophagy proteins, it possesses a putative phospholipid scramblase, TgATG9. Through structural modeling and complementation assays, we show herein that TgATG9 can partially rescue bulk autophagy in atg9∆ yeast. We demonstrated the importance of TgATG9 for proper autophagosome dynamics at the subcellular level using three-dimensional live cell lattice light sheet microscopy. Conditional knockdown of TgATG9 in T. gondii after bradyzoite differentiation resulted in markedly reduced parasite viability. Together, our findings provide insights into the molecular dynamics of autophagosome biogenesis within an early-branching eukaryote and pinpoint the indispensable role of autophagy in maintaining T. gondii chronic infection.

This study investigates the molecular prevalence and phylogenetic characteristics of two prominent blood-borne pathogens, Toxoplasma gondii (T. gondii) and Plasmodium spp., in common quails (Coturnix coturnix) sampled from both wild (N = 236) and farmed (N = 197) populations across four districts (Layyah, Dera Ghazi Khan, Lahore, and Multan) in Punjab, Pakistan, during the hunting seasons from 2021 to 2023. Additionally, the impact of these pathogens on the complete blood count (CBC) of the hosts is examined. Out of 433 quails tested, 25 (5.8%) exhibited amplification of the internal transcribed spacer (ITS-1) gene for T. gondii, while 15 (3.5%) showed amplification of the Cytochrome b gene for Plasmodium spp. A risk factor analysis indicated that the prevalence of both pathogens was not confined to specific sampling sites or bird sexes (P > 0.05). District-wise analysis highlighted that hens were more susceptible to both T. gondii and Plasmodium spp. infections than cocks. Wild quails exhibited a higher susceptibility to T. gondii compared to farmed birds. Significant CBC variations were recorded in infected birds as compared to uninfected ones. BLAST analysis of generated sequences has confirmed the identity of recovered PCR amplicons as T. gondii and Plasmodium relictum. Phylogenetic analysis revealed that Pakistani isolates clustered with those reported from various countries globally. This study provides the first documentation of T. gondii and Plasmodium sp. infections in Pakistani quails, underscoring the need for detailed investigations across different regions to enhance our understanding of infection rates and the zoonotic potential of these parasites. [ABSTRACT FROM AUTHOR]

Microsporidia and Apicomplexa are eukaryotic, single‐celled, intracellular parasites with huge public health and economic importance. Typically, these parasites are studied separately, emphasizing their uniqueness and diversity. In this review, we explore the huge amount of genomic data that has recently become available for the two groups. We compare and contrast their genome evolution and discuss how their transitions to intracellular life may have shaped it. In particular, we explore genome reduction and compaction, genome expansion and ploidy, gene shuffling and rearrangements, and the evolution of centromeres and telomeres. [ABSTRACT FROM AUTHOR]

Apicomplexan parasites harbor a complex endomembrane system as well as unique secretory organelles. These complex cellular structures require an elaborate vesicle trafficking system, which includes Rab GTPases and their regulators, to assure the biogenesis and secretory of the organelles. Here we exploit the model apicomplexan organism Toxoplasma gondii that encodes a family of Rab GTPase Activating Proteins, TBC (Tre-2/Bub2/Cdc16) domain-containing proteins. Functional profiling of these proteins in tachyzoites reveals that TBC9 is the only essential regulator, which is localized to the endoplasmic reticulum (ER) in T. gondii strains. Detailed analyses demonstrate that TBC9 is required for normal distribution of proteins targeting to the ER, and the Golgi apparatus in the parasite, as well as for the normal formation of daughter inner membrane complexes (IMCs). Pull-down assays show a strong protein interaction between TBC9 and specific Rab GTPases (Rab11A, Rab11B, and Rab2), supporting the role of TBC9 in daughter IMC formation and early vesicular transport. Thus, this study identifies the only essential TBC domain-containing protein TBC9 that regulates early vesicular transport and IMC formation in T. gondii and potentially in closely related protists. A study suggests that TBC9, one of the Rab GTPase Activating Proteins, is crucial for early vesicular transport and inner membrane complex formation by regulating Rab2, Rab11A and Rab11B in Toxoplasma gondii. [ABSTRACT FROM AUTHOR]

The discovery of a relict plastid, also known as an apicoplast (apicomplexan plastid), that houses housekeeping processes and metabolic pathways critical to Plasmodium parasites' survival has prompted increased research on identifying potent inhibitors that can impinge on apicoplast-localised processes. The apicoplast is absent in humans, yet it is proposed to originate from the eukaryote's secondary endosymbiosis of a primary symbiont. This symbiotic relationship provides a favourable microenvironment for metabolic processes such as haem biosynthesis, Fe-S cluster synthesis, isoprenoid biosynthesis, fatty acid synthesis, and housekeeping processes such as DNA replication, transcription, and translation, distinct from analogous mammalian processes. Recent advancements in comprehending the biology of the apicoplast reveal it as a vulnerable organelle for malaria parasites, offering numerous potential targets for effective antimalarial therapies. We provide an overview of the metabolic processes occurring in the apicoplast and discuss the organelle as a viable antimalarial target in light of current advances in drug discovery. We further highlighted the relevance of these metabolic processes to Plasmodium falciparum during the different stages of the lifecycle. [ABSTRACT FROM AUTHOR]

Infection with the apicomplexan protozoan Toxoplasma gondii can be life-threatening in immunocompromised hosts. Transmission frequently occurs through the oral ingestion of T. gondii bradyzoite cysts, which transition to tachyzoites, disseminate, and then form cysts containing bradyzoites in the central nervous system, resulting in latent infection. Encapsulation of bradyzoites by a cyst wall is critical for immune evasion, survival, and transmission. O-glycosylation of the protein CST1 by the mucin-type O-glycosyltransferase T. gondii (Txg) GalNAc-T3 influences cyst wall rigidity and stability. Here, we report X-ray crystal structures of TxgGalNAc-T3, revealing multiple features that are strictly conserved among its apicomplexan homologues. This includes a unique 2nd metal that is coupled to substrate binding and enzymatic activity in vitro and cyst wall O-glycosylation in T. gondii. The study illustrates the divergence of pathogenic protozoan GalNAc-Ts from their host homologues and lays the groundwork for studying apicomplexan GalNAc-Ts as therapeutic targets in disease. A Toxoplasma gondii mucin-type O-glycosyltransferase uses a unique catalytic mechanism to modify bradyzoite cyst wall proteins. A second metal coupled to substrate binding is required for catalysis, while an active site glutamate suggests a double-displacement mechanism. [ABSTRACT FROM AUTHOR]

Coccidiosis is an intestinal protozoan disease that affects the poultry industry worldwide. The severity of this disease varies depending on the identity of the infectious agents. Therefore, this study was carried out to identify the Eimeria species that affect broiler chickens, Gallus gallus domesticus, through morphological and molecular phylogenetic analyses. Twenty‐five faecal samples were collected from the broiler chickens in a commercial poultry farm in Riyadh (Saudi Arabia). Using the floatation technique, faeces were examined microscopically for the Eimeria occurrence. Identification of Eimeria species was performed based on morphological criteria and molecular tools (DNA amplification for the partial small subunit ribosomal RNA (18S rRNA), internal transcribed spacer (ITS)‐1, and mitochondrial cytochrome c oxidase I (COI) genes. In this study, 32% (8 out of 25) of collected samples were found to be positive for coccidiosis. After sporulation in potassium dichromate (K2Cr2O7), the sporulated oocysts were observed as ovoid and measured 18.37–23.19 µm (19.87) long and 15.07–18.67 µm (16.46) wide, with the anterior location of a polar granule and absence of micropyle. These Eimeria oocysts were assumed to size and shape characteristics of Eimeria acervulina. Molecular analysis was conducted on the sequences of the polymerase chain reaction products from the three genes studied (18S rRNA, ITS‐1, and COI). At the three genes, results showed that the resultant sequences clustered with E. acervulina from different regions confirming morphological description. This study highlighted the importance of molecular techniques to detect avian Eimeria species more than the traditional morphology‐based tool to optimise the appropriate anticoccidial strategies for long‐term control in the studied area. [ABSTRACT FROM AUTHOR]

The micropore contains two distinct morphological types, with type I being present in most apicomplexans. The micropore is a functional nutrient uptake organelle and is involved in artemisinin resistance in Plasmodium. Although possessing distinct morphologies, type I and II micropores share a core set of components across apicomplexan species. A new model for micropore assembly is proposed which involves sequential recruitment of EPS15 (epidermal growth factor receptor substrate-15), Kelch13, and UBP1 (ubiquitin binding protein-1). Future studies should consider the transformation between type I and type II micropores. The micropore, a mysterious structure found in apicomplexan species, was recently shown to be essential for nutrient acquisition in Plasmodium falciparum and Toxoplasma gondii. However, the differences between the micropores of these two parasites questions the nature of a general apicomplexan micropore structure and whether the formation process model from Plasmodium can be applied to other apicomplexans. We analyzed the literature on different apicomplexan micropores and found that T. gondii probably harbors a more representative micropore type than the more widely studied ones in Plasmodium. Using recent knowledge of the Kelch 13 (K13) protein interactome and gene depletion phenotypes in the T. gondii micropore, we propose a model of micropore formation, thus enriching our wider understanding of micropore protein function. [ABSTRACT FROM AUTHOR]

The subpellicular microtubules (SPMTs) shape the parasite, contributing to its motility and adaptation across various environments. They possess a high density of microtubule (MT)-associated proteins, providing exceptional stability. The conoid consists of tubulin fibers uniquely shaped by MT-associated proteins. Conoid extrusion through the apical polar ring regulates parasite motility by acting as a gatekeeper, controlling F-actin access to the intrapellicular space. The intraconoidal MTs (ICMTs) play a pivotal role in positioning the rhoptries, facilitating their iterative discharge to enable successful invasion and subversion of host cellular functions. The role of kinesins and dyneins in trafficking and MT dynamics remains understudied in Toxoplasma gondii , a parasite that also has a yet-to-be-deciphered tubulin code, which defines MT properties. Microtubules (MTs) play a vital role as key components of the eukaryotic cytoskeleton. The phylum Apicomplexa comprises eukaryotic unicellular parasitic organisms defined by the presence of an apical complex which consists of specialized secretory organelles and tubulin-based cytoskeletal elements. One apicomplexan parasite, Toxoplasma gondii , is an omnipresent opportunistic pathogen with significant medical and veterinary implications. To ensure successful infection and widespread dissemination, T. gondii heavily relies on the tubulin structures present in the apical complex. Recent advances in high-resolution imaging, coupled with reverse genetics, have offered deeper insights into the composition, functionality, and dynamics of these tubulin-based structures. The apicomplexan tubulins differ from those of their mammalian hosts, endowing them with unique attributes and susceptibility to specific classes of inhibitory compounds. [ABSTRACT FROM AUTHOR]

The cytoskeletal organization of a squirmid, namely Platyproteum vivax, was investigated with confocal laser scanning microscopy (CLSM) to refine inferences about convergent evolution among intestinal parasites of marine invertebrates. Platyproteum inhabits Pacific peanut worms (Phascolosoma agassizii) and has traits that are similar to other lineages of myzozoan parasites, namely gregarine apicomplexans within Selenidium, such as conspicuous feeding stages, called "trophozoites," capable of dynamic undulations. SEM and CLSM of P. vivax revealed an inconspicuous flagellar apparatus and a uniform array of longitudinal microtubules organized in bundles (LMBs). Extreme flattening of the trophozoites and a consistently oblique morphology of the anterior end provided a reliable way to distinguish dorsal and ventral surfaces. CLSM revealed a novel system of microtubules oriented in the flattened dorsoventral plane. Most of these dorsoventral microtubule bundles (DVMBs) had a punctate distribution and were evenly spaced along a curved line spanning the longitudinal axis of the trophozoites. This configuration of microtubules is inferred to function in maintaining the flattened shape of the trophozoites and facilitate dynamic undulations. The novel traits in Platyproteum are consistent with phylogenomic data showing that this lineage is only distantly related to Selenidium and other marine gregarine apicomplexans with dynamic intestinal trophozoites. [ABSTRACT FROM AUTHOR]

Four new wood-inhabiting fungi, Trechispora albofarinosa, T. bisterigmata, T. pileata and T. wenshanensis spp. nov., are proposed based on a combination of morphological features and molecular evidence. Trechispora albofarinosa is characterized by the farinose basidiomata with flocculence hymenial surface, a monomitic hyphal system with clamped generative hyphae, and ellipsoid, warted basidiospores. Trechispora bisterigmata is characterized by the membranous basidiomata with odontioid hymenial surface, rhizomorphic sterile margin, barrelled basidia and subglobose to broad ellipsoid, smooth basidiospores. Trechispora pileata is characterized by the laterally contracted base, solitary or imbricate basidiomata, fan shaped pileus, radially striate-covered surface with appressed scales, odontioid hymenophore surface, and subglobose to broad ellipsoid, thin-walled, smooth basidiospores. Trechispora wenshanensis is characterized by a cottony basidiomata with a smooth hymenial surface, and ellipsoid, thin-walled, warted basidiospores. Sequences of ITS and LSU marker of the studied samples were generated, and phylogenetic analyses were performed with the maximum likelihood, maximum parsimony, and Bayesian inference methods. The phylogenetic tree inferred from the ITS+nLSU sequences highlighted that four new species were grouped into the genus Trechispora. [ABSTRACT FROM AUTHOR]

The use of fluorescent proteins (FPs) in Plasmodium parasites has been key to understand the biology of this obligate intracellular protozoon. FPs like the green fluorescent protein (GFP) enabled to explore protein localization, promoter activity as well as dynamic processes like protein export and endocytosis. Furthermore, FP biosensors have provided detailed information on physiological parameters at the subcellular level, and fluorescent reporter lines greatly extended the malariology toolbox. Still, in order to achieve optimal results, it is crucial to know exactly the properties of the FP of choice and the genetic scenario in which it will be used. This review highlights advantages and disadvantages of available landing sites and promoters that have been successfully applied for the ectopic expression of FPs in Plasmodium berghei and Plasmodium falciparum. Furthermore, the properties of newly developed FPs beyond DsRed and EGFP, in the visualization of cells and cellular structures as well as in the sensing of small molecules are discussed. [ABSTRACT FROM AUTHOR]

This study aims to determine the prevalence of Cryptosporidium and Eimeria spp. oocysts in fish specimens in the river Kura. It was conducted during the 2021-2022 at two sites: Mingachevir reservoir in central Azerbaijan and in Neftchala district where the river finally enters the Caspian Sea through a delta of the Kura River estuary. The diagnosis of oocysts was performed microscopically. Fine smears from the intestine epithelial layers stained by Ziehl-Neelsen for Cryptosporidium oocysts. To identify Eimeria oocysts, each fish’s faecal material and intestinal scrapings were examined directly under a light microscope in wet samples on glass slides with a coverslip. Results revealed a prevalence of Cryptosporidium and Eimeria species infections in fish hosts from both territories Rutilus caspicus, Alburnus filippi, Abramis brama orientalis and Carassius gibelio. Of 170 investigated fish specimens, 8.8% (15/170) were infected with Cryptosporidium species oocysts. Eimeria species oocysts were identified in 20.6% (35/170). The presence of Cryptosporidium and Eimeria infections in fish specimens are natural infections. However, their presence in fish species may be attributed to the age of the fish species and water pollution. This is the first report regarding the prevalence of Cryptosporidium oocysts in fish species in Azerbaijan. [ABSTRACT FROM AUTHOR]

Hepatozoon species (phylum Apicomplexa: Adeleorina, Hepatozooidae) are the cause of canine hepatozoonosis, a vector-borne disease (VBD) spread by ticks (Ixodidae). The aim of the present study was to determine the prevalence of Hepatozoon canis (H. canis) infections in different pet dogs admitted to different clinics in Egypt as well as determine the effect of H. canis on dogs through analysis of the immunogenic genes and the oxidative stress markers. One hundred dogs with different clinical signs, fever, fatigue, tick infestation around the ears and neck region, were examined. Thin blood films were performed and stained with Giemsa. Thirty examined dogs (30%) were positive for H. canis by direct observation of the gamonts in circulating leucocytes on blood smears stained with Giemsa. Two hundred ticks were identified morphologically and all ticks were classified as R. sanguineus. Hematological and biochemical results of sampled dogs were recorded. The AST and ALT levels were higher than control-negative healthy dogs. MDA levels in H. canis infected dogs were higher than that of control negative dogs. The transcript levels of the different targeted genes (IL-1β; IL6; TNF-α and IFN-γ) were upregulated in infected dogs with H. canis significantly than control healthy dogs. Canine hepatozoonosis induced tissue reaction evaluated by different immunological genes and oxidative stress. [ABSTRACT FROM AUTHOR]

Apicomplexan parasites are aetiological agents of numerous diseases in humans and livestock. Functional genomics studies in these parasites enable the identification of biological mechanisms and protein functions that can be targeted for therapeutic intervention. Recent improvements in forward genetics and whole‐genome screens utilising CRISPR/Cas technology have revolutionised the functional analysis of genes during Apicomplexan infection of host cells. Here, we highlight key discoveries from CRISPR/Cas9 screens in Apicomplexa or their infected host cells and discuss remaining challenges to maximise this technology that may help answer fundamental questions about parasite–host interactions. [ABSTRACT FROM AUTHOR]

Apicomplexan parasites comprise significant pathogens of humans, livestock and wildlife, but also represent a diverse group of eukaryotes with interesting and unique cell biology. The study of cell biology in apicomplexan parasites is complicated by their small size, and historically this has required the application of cutting‐edge microscopy techniques to investigate fundamental processes like mitosis or cell division in these organisms. Recently, a technique called expansion microscopy has been developed, which rather than increasing instrument resolution like most imaging modalities, physically expands a biological sample. In only a few years since its development, a derivative of expansion microscopy known as ultrastructure‐expansion microscopy (U‐ExM) has been widely adopted and proven extremely useful for studying cell biology of Apicomplexa. Here, we review the insights into apicomplexan cell biology that have been enabled through the use of U‐ExM, with a specific focus on Plasmodium, Toxoplasma and Cryptosporidium. Further, we summarize emerging expansion microscopy modifications and modalities and forecast how these may influence the field of parasite cell biology in future. [ABSTRACT FROM AUTHOR]