A few specialized tick-transmitted protozoa cause fatal infectious diseases in cattle through a unique cell biological mechanism, the transformation of host leukocytes into cancer-like cells. These ‘transformative’ parasites, such as Theileria annulata, hijack host signaling pathways and, during the schizont stage of their complex life cycle, cause uncontrolled proliferation and immortalization of infected cells, likely through a broad repertoire of fine-tuned effector proteins. This review summarizes recent advancements on the parasite-host interactions proposed to underlie this remarkable transformation phenotype. Exported parasite protein families are significantly expanded in T. annulata and often exhibit intrinsically disordered protein regions as a consistent motif. We highlight current knowledge gaps and technical challenges, whose resolution promises a better understanding of parasite effector function. Expanding our understanding of this exceptional eukaryotic system could not only enhance strategies for controlling disease but also provide fundamental insights into cell and cancer biology.

The probability that a disease will manifest is highly variable. Susceptibility to disease is influenced by genetic background, environment and lifestyle choices. In this review, we put forward the premise that evolution of disease susceptibility may be partially influenced by the interaction of divergent pathogen DNA-binding proteins with variable binding sites in the host genome. The hypothesis put forward is derived from recent data obtained from work on the protozoan parasite, Theileria annulata , together with related evidence from viral and bacterial pathogens that have been postulated to modulate host epigenome architecture. The pathogen proteins highlighted have the potential to mimic functions of mammalian epigenome organisers linked to a range of disease syndromes. It is feasible, therefore, that the evolutionary relationship between pathogen and host impacts susceptibility to a range of conditions, such as autoimmune disorders and cancer, which are not directly linked to pathogen infection.

Parasitic diseases, particularly malaria (caused by Plasmodium falciparum) and theileriosis (caused by Theileria spp.), profoundly impact global health and the socioeconomic well-being of lower-income countries. Despite recent advances, identifying host metabolic proteins essential for these auxotrophic pathogens remains challenging. Here, we generate a novel metabolic model of human hepatocytes infected with P. falciparum and integrate it with a genome-wide CRISPR knockout screen targeting Theileria-infected cells to pinpoint shared vulnerabilities. We identify key host metabolic enzymes critical for the intracellular survival of both of these lethal hemoparasites. Remarkably, among the metabolic proteins identified by our synergistic approach, we find that host purine and heme biosynthetic enzymes are essential for the intracellular survival of P. falciparum and Theileria, while other host enzymes are only essential under certain metabolic conditions, highlighting P. falciparum's adaptability and ability to scavenge nutrients selectively. Unexpectedly, host porphyrins emerge as being essential for both parasites. The shared vulnerabilities open new avenues for developing more effective therapies against these debilitating diseases, with the potential for broader applicability in combating apicomplexan infections.

ABSTRACT Theileria annulata is a tick-transmitted apicomplexan parasite that gained the unique ability among parasitic eukaryotes to transform its host cell, inducing a fatal cancer-like disease in cattle. Understanding the mechanistic interplay between the host cell and malignant Theileria species that drives this transformation requires the identification of responsible parasite effector proteins. In this study, we used TurboID-based proximity labeling, which unbiasedly identified secreted parasite proteins within host cell compartments. By fusing TurboID to nuclear export or localization signals, we biotinylated proteins in the vicinity of the ligase enzyme in the nucleus or cytoplasm of infected macrophages, followed by mass spectrometry analysis. Our approach revealed with high confidence nine nuclear and four cytosolic candidate parasite proteins within the host cell compartments, eight of which had no orthologs in non-transforming T. orientalis. Strikingly, all eight of these proteins are predicted to be highly intrinsically disordered proteins. We discovered a novel tandem arrayed protein family, nuclear intrinsically disordered proteins (NIDP) 1–4, featuring diverse functions predicted by conserved protein domains. Particularly, NIDP2 exhibited a biphasic host cell-cycle-dependent localization, interacting with the EB1/CD2AP/CLASP1 parasite membrane complex at the schizont surface and the tumor suppressor stromal antigen 2 (STAG2), a cohesion complex subunit, in the host nucleus. In addition to STAG2, numerous NIDP2-associated host nuclear proteins implicated in various cancers were identified, shedding light on the potential role of the T. annulata exported protein family NIDP in host cell transformation and cancer-related pathways.IMPORTANCETurboID proximity labeling was used to identify secreted proteins of Theileria annulata, an apicomplexan parasite responsible for a fatal, proliferative disorder in cattle that represents a significant socio-economic burden in North Africa, central Asia, and India. Our investigation has provided important insights into the unique host-parasite interaction, revealing secreted parasite proteins characterized by intrinsically disordered protein structures. Remarkably, these proteins are conspicuously absent in non-transforming Theileria species, strongly suggesting their central role in the transformative processes within host cells. Our study identified a novel tandem arrayed protein family, with nuclear intrinsically disordered protein 2 emerging as a central player interacting with established tumor genes. Significantly, this work represents the first unbiased screening for exported proteins in Theileria and contributes essential insights into the molecular intricacies behind the malignant transformation of immune cells.

SUMMARY Apicomplexan parasite diseases, including malaria ( Plasmodium ) and theileriosis ( Theileria ), pose a significant threat to global health and the socioeconomic well-being of low-income countries. Despite recent advances, the common host metabolic proteins essential for these highly auxotrophic pathogens remain elusive. Here, we present a comprehensive investigation integrating a metabolic model of P. falciparum parasites in hepatocytes and a genome-wide CRISPR screen targeting Theileria schizont-infected macrophages. We reveal unifying host metabolic enzymes critical for the intracellular survival of these related hematozoa. We show that pathways such as host purine and heme biosynthesis are essential for both Theileria survival and Plasmodium liver development, while genes involved in glutathione and polyamine biosynthesis are predicted to be essential for Plasmodium only under certain metabolic conditions. Our work highlights the importance of host porphyrins for the viability of liver-stage Plasmodium . Shared parasite vulnerabilities provide a resource for exploring alternative therapeutic approaches to combat these crippling diseases.

Intracellular pathogens construct their environmental niche, and influence disease susceptibility, by deploying factors that manipulate infected host cell gene expression. Theileria annulata is an important tick-borne parasite of cattle that causes tropical theileriosis. Excellent candidates for modulating host cell gene expression are DNA binding proteins bearing AT-hook motifs encoded within the TashAT gene cluster of the parasite genome. In this study, TashAT2 was transfected into bovine BoMac cells to generate three expressing and three non-expressing (opposite orientation) cell lines. RNA-Seq was conducted and differentially expressed (DE) genes identified. The resulting dataset was compared with genes differentially expressed between infected cells and non-infected cells, and DE genes between infected cell lines from susceptible Holstein vs tolerant Sahiwal cattle. Over 800 bovine genes displayed differential expression associated with TashAT2, 209 of which were also modulated by parasite infection. Network analysis showed enrichment of DE genes in pathways associated with cellular adhesion, oncogenesis and developmental regulation by mammalian AT-hook bearing high mobility group A (HMGA) proteins. Overlap of TashAT2 DE genes with Sahiwal vs Holstein DE genes revealed that a significant number of shared genes were associated with disease susceptibility. Altered protein levels encoded by one of these genes (GULP1) was strongly linked to expression of TashAT2 in BoMac cells and was demonstrated to be higher in infected Holstein leucocytes compared to Sahiwal. We conclude that TashAT2 operates as an HMGA analogue to differentially mould the epigenome of the infected cell and influence disease susceptibility.

BACKGROUND:Cryptosporidiosis is a parasitic disease associated with potentially fatal diarrhea. The most used method in Cryptosporidium subtyping is based on the glycoprotein gene gp60. Each infection can represent a parasite population, and it is important to investigate the influence on transmission and virulence, as well as any impact on public health investigations. However, an easy-to-use method for detection is lacking.METHODS:Here we report on the use of the bioinformatic program TIDE for deconvolution of gp60 chromatograms. A combination of single oocyst analysis and cloning successfully confirmed the within-sample parasite population diversity. Retrospective sample analysis was conducted on archived chromatograms.RESULTS:For Cryptosporidium parvum, 8.6% multistrain infections (13 of 152) obscured by currently used consensus base calling were detected. Importantly, we show that single oocysts can harbor a mixed population of sporozoites. We also identified a striking dominance of unappreciated polymerase stutter artefacts in all 218 chromatograms analyzed, challenging the uncritical use of gp60 typing.CONCLUSIONS:We demonstrate the value of a new, easy-to-use analytical procedure for critical characterization of C. parvum and Cryptosporidium hominis in epidemiological investigations, also applicable retrospectively. Our findings illuminate the hidden parasite diversity with important implications for tracing zoonotic and person-to-person transmissions.

Infections with intravascular digenean trematodes of the Spirorchiidae family (spirorchiidoses) are of great conservation concern both in marine and freshwater turtles due to their pathogenic potential. Between 2014 and 2021, Spirorchis sp. infections associated with granulomatous inflammation and sudden death were detected in European pond turtles (Emys orbicularis) from three conservation breeding facilities in Switzerland. Blood fluke eggs associated with lesions were found in the intestine, spleen, testis, skeletal musculature, heart, kidneys, stomach, pancreas, liver, lung, and meninges from nine pond turtles submitted for necropsy and in the intestinal content from five of these animals. Two novel polymerase chain reactions (PCRs) targeting the 28S ribosomal RNA gene and the ITS2 region and subsequent sequencing revealed 100% nucleotide identity with a Spirorchis sp. previously isolated from an Escambia map turtle (Graptemys ernsti) in the USA. Our findings suggest a spill-over event secondary to direct or indirect contact with invasive North American turtle species in Switzerland. We describe the clinical, haematological, ultrasonographical, endoscopical, parasitological, pathological, and molecular findings associated with spirorchiid blood fluke infections of the Spirorchis genus in E. orbicularis, as well as the biosecurity measures that were developed to prevent the spread of this parasite among breeding and highly endangered free-ranging E. orbicularis populations in Switzerland.

Herein, we developed a single and a duplex TaqMan quantitative PCR (qPCR) for absolute quantification of copy numbers of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker for pyrimethamine resistance in Toxoplasma gondii knockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the inserted mdhfr-ts and of the wild-type (WT) dhfr-ts (wtdhfr-ts) which is present as single copy gene in Toxoplasma and encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of the dhfr-ts fragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number of dhfr-ts copies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bp dhfr-ts fragment and the T. gondii 529-bp repeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given by dhfr-ts amplification is equal to the number of tachyzoites determined by amplification of the Toxoplasma 529-bp, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration of mdhfr-ts, the calculated ratio is 2. We then applied both approaches to test T. gondii RH mutants in which the major surface antigen (SAG1) was disrupted through insertion of mdhfr-ts using CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integrated mdhfr-ts. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic of T. gondii KOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions of mdhfr-ts. The principle of the duplex TaqMan qPCR is applicable for other selectable markers in Toxoplasma. TaqMan qPCR tools may contribute to more frequent use of WT Toxoplasma strains during functional genomics.

Conservation of endangered animal species is a major task of zoos. Husbandry and breeding of Atlantic puffins Fratercula arctica in captivity is challenging. In 2019, the entire chick population (n = 4 chicks) in Berne Animal Park's Atlantic puffin colony (Bern, Switzerland) died within 7 d. Due to supply constraints, the chicks had been fed with wild-caught European minnows Phoxinus phoxinus. At necropsy, the main pathological finding in all deceased puffin chicks was a multifocal, moderate to severe subacute heterophilic and granulomatous enteritis with intralesional adult trematodes and eggs. Metacercariae surrounded by few necrotic cells and scattered macrophages were found in the brain and spinal cord of the food fish. Additional microbiological analyses of both the puffin chicks and fish were unremarkable. Diplostomum phoxini DNA could be identified in formalin-fixed paraffin-embedded tissue from the small intestine of all puffin chicks and European minnows following PCR and sequencing of the 18S ribosomal RNA gene and the internal transcribed spacer (ITS1) region. This report illustrates the importance of intensive health checks of food fish for animal species kept in captivity.

Oral infection with Toxoplasma gondii results in dysbiosis and enteritis, both of which revert to normal during chronic infection. However, whether infection leaves a lasting impact on mucosal responses remains uncertain. Here we examined the effect of the chemical irritant dextran sodium sulfate (DSS) on intestinal damage and wound healing in chronically infected mice. Our findings indicate that prior infection with T. gondii exacerbates damage to the colon caused by DSS and impairs wound healing by suppressing stem cell regeneration of the epithelium. Enhanced tissue damage was attributable to inflammatory monocytes that emerge preactivated from bone marrow, migrate to the intestine, and release inflammatory mediators, including nitric oxide. Tissue damage was reversed by neutralization of inflammatory monocytes or nitric oxide, revealing a causal mechanism for tissue damage. Our findings suggest that chronic infection with T. gondii enhances monocyte activation to increase inflammation associated with a secondary environmental insult.

A juvenile Little Owl (Athene noctua) was diagnosed with granulomatous encephalitis and muscular sarcocysts. Sarcocystis halieti was identified in the brain and muscle tissue by PCR and subsequent sequencing. This is the first report of S. halieti as a potential encephalitis-causing pathogen in birds.

One of the first events that follows invasion of leukocytes by Theileria sporozoites is the destruction of the surrounding host cell membrane and the rapid association of the intracellular parasite with host microtubules. This is essential for the parasite to establish its niche within the cytoplasm of the invaded leukocyte and sets Theileria spp. apart from other members of the apicomplexan phylum such as Toxoplasma gondii and Plasmodium spp., which reside within the confines of a host-derived parasitophorous vacuole. After establishing infection, transforming Theileria species (T. annulata, T. parva) significantly rewire the signaling pathways of their bovine host cell, causing continual proliferation and resistance to ligand-induced apoptosis, and conferring invasive properties on the parasitized cell. Having transformed its target cell, Theileria hijacks the mitotic machinery to ensure its persistence in the cytoplasm of the dividing cell. Some of the parasite and bovine proteins involved in parasite-microtubule interactions have been fairly well characterized, and the schizont expresses at least two proteins on its membrane that contain conserved microtubule binding motifs. Theileria-encoded proteins have been shown to be translocated to the host cell cytoplasm and nucleus where they have the potential to directly modify signaling pathways and host gene expression. However, little is known about their mode of action, and even less about how these proteins are secreted by the parasite and trafficked to their target location. In this review we explore the strategies employed by Theileria to transform leukocytes, from sporozoite invasion until immortalization of the host cell has been established. We discuss the recent description of nuclear pore-like complexes that accumulate on membranes close to the schizont surface. Finally, we consider putative mechanisms of protein and nutrient exchange that might occur between the parasite and the host. We focus in particular on differences and similarities with recent discoveries in T. gondii and Plasmodium species.

Toxoplasma gondii and Hammondia hammondi are closely-related coccidian intracellular parasites that differ in their ability to cause disease in animal and (likely) humans. The role of the host response in these phenotypic differences is not known and to address this we performed a transcriptomic analysis of a monocyte cell line (THP-1) infected with these two parasite species. The pathways altered by infection were shared between species ~95% the time, but the magnitude of the host response to H. hammondi was significantly higher compared to T. gondii. Accompanying this divergent host response was an equally divergent impact on the cell cycle of the host cell. In contrast to T. gondii, H. hammondi infection induces cell cycle arrest via pathways linked to DNA-damage responses and cellular senescence and robust secretion of multiple chemokines that are known to be a part of the senescence associated secretory phenotype (SASP). Remarkably, prior T. gondii infection or treatment with T. gondii-conditioned media suppressed responses to H. hammondi infection, and promoted the replication of H. hammondi in recipient cells. Suppression of inflammatory responses to H. hammondi was found to be mediated by the T. gondii effector IST, and this finding was consistent with reduced functionality of the H. hammondi IST ortholog compared to its T. gondii counterpart. Taken together our data suggest that T. gondii manipulation of the host cell is capable of suppressing previously unknown stress and/or DNA-damage induced responses that occur during infection with H. hammondi, and that one important impact of this T. gondii mediated suppression is to promote parasite replication.

BackgroundOsteomyelitis is relatively frequent in young pigs and a few bacterial species have been postulated to be potential causative agents. Although Actinobacillus (A.) pleuropneumoniae has been sporadically described to cause osteomyelitis, typically, actinobacillosis is characterized by respiratory symptoms. Nevertheless, subclinical infections are a challenging problem in pig herds. To the authors' knowledge, this is the first case description that reports clinical, diagnostic imaging, pathological and histopathological findings of vertebral osteomyelitis in a pig and first describes A. pleuropneumoniae as the causative agent identified by advanced molecular methods.Case presentationAn eight-week-old female weaner was presented with a non-ambulatory tetraparesis. The neurological signs were consistent with a lesion in the C6-T2 spinal cord segments. Imaging studies revealed a collapse of the seventh cervical vertebral body (C7) with a well demarcated extradural space-occupying mass ventrally within the vertebral canal severely compressing the spinal cord. Post-mortem examination identified an abscess and osteomyelitis of C7 and associated meningitis and neuritis with subsequent pathological fracture of C7 and compression of the spinal cord. In the microbiological analysis, A. pleuropneumoniae was identified using PCR and DNA sequence analysis.ConclusionsA. pleuropneumoniae can be responsible for chronic vertebral abscess formation with subsequent pathological fracture and spinal cord compression in pigs.

The canid tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE) in humans and other intermediate hosts. Depending on the permissiveness of the intermediate host, the larval form of E. multilocularis (metacestode) may be either fertile, e.g. in rodents, and thus supporting the life cycle of the parasite, or infertile, e.g. in pigs, and thus interrupting the life cycle. Pigs have been shown to act as aberrant hosts for the metacestode and consequently develop liver lesions but represent a dead-end for the parasite. Routine liver inspection at slaughter provided the basis for a large-scale surveillance study on E. multilocularis infection in pigs. The aim of this combined cross-sectional and case-control study was to estimate the minimal prevalence of E. multilocularis in pigs in Switzerland, to find factors associated with infection, and to assess potential regional clusters of infection. During the 12-month-study period, approximately 85% of all pigs slaughtered in Switzerland were assessed. In total, 450 pig livers with macroscopic lesions suggestive of E. multilocularis infection were analysed. Of those, 200 samples were positive by E. multilocularis-PCR. Thus, the overall minimal prevalence detected by molecular means was 0.009% in all slaughter pigs (200 of 2'143'996), 0.008% in finishing pigs (177 of 2'123'542), and 0.11% in breeding pigs (22 of 20'454). Histology revealed the unique presence of a laminated layer in 105 cases, and an additional germinal layer detected in a single case. Protoscoleces could not be observed in any of the lesions. Factors positively associated with infection were "foxes seen in the pig shed", "foxes on premises", "presence of other animals in the shed", "absence of a hygiene barrier", "outdoor feeding", "feeding grass", "lack of rodent control", "not having own dogs on the farm" and "infrequent deworming of sows". Infection was present in all regions sampled and was representative of the important pig rearing areas of Switzerland, without evidence of any obvious geographical cluster. Conclusively, our study provided further evidence of widespread environmental contamination with E. multilocularis eggs in Switzerland. Furthermore, the absence of protoscoleces in any of the lesions supported the concept that pigs act only as a dead-end host and thus do not contribute to the life cycle of the parasite. Factors associated with E. multilocularis infection were in-line with parasite biology, and many can be addressed by increasing hygiene and management standards.