Intracellular single-celled parasites belonging to the large phylum Apicomplexa are amongst the most prevalent and morbidity-causing pathogens worldwide. In this review, we highlight a few of the many recent advances in the field that helped to clarify some important aspects of their fascinating biology and interaction with their hosts. Plasmodium falciparum causes malaria, and thus the recent emergence of resistance against the currently used drug combinations based on artemisinin has been of major interest for the scientific community. It resulted in great advances in understanding the resistance mechanisms that can hopefully be translated into altered future drug regimens. Apicomplexa are also experts in host cell manipulation and immune evasion. Toxoplasma gondii and Theileria sp., besides Plasmodium sp., are species that secrete effector molecules into the host cell to reach this aim. The underlying molecular mechanisms for how these proteins are trafficked to the host cytosol (T. gondii and Plasmodium) and how a secreted protein can immortalize the host cell (Theileria sp.) have been illuminated recently. Moreover, how such secreted proteins affect the host innate immune responses against T. gondii and the liver stages of Plasmodium has also been unraveled at the genetic and molecular level, leading to unexpected insights. Methodological advances in metabolomics and molecular biology have been instrumental to solving some fundamental puzzles of mitochondrial carbon metabolism in Apicomplexa. Also, for the first time, the generation of stably transfected Cryptosporidium parasites was achieved, which opens up a wide variety of experimental possibilities for this understudied, important apicomplexan pathogen.

Highly charged compounds typically suffer from low membrane permeability and thus are generally regarded as sub-optimal drug candidates. Nonetheless, the highly charged drug fosmidomycin and its more active methyl-derivative FR900098 have proven parasiticidal activity against erythrocytic stages of the malaria parasite Plasmodium falciparum. Both compounds target the isoprenoid biosynthesis pathway present in bacteria and plastid-bearing organisms, like apicomplexan parasites. Surprisingly, the compounds are inactive against a range of apicomplexans replicating in nucleated cells, including Toxoplasma gondii. Since non-infected erythrocytes are impermeable for FR90098, we hypothesized that these drugs are taken up only by erythrocytes infected with Plasmodium. We provide evidence that radiolabeled FR900098 accumulates in theses cells as a consequence of parasite-induced new properties of the host cell, which coincide with an increased permeability of the erythrocyte membrane. Babesia divergens, a related parasite that also infects human erythrocytes and is also known to induce an increase in membrane permeability, displays a similar susceptibility and uptake behavior with regard to the drug. In contrast, Toxoplasma gondii-infected cells do apparently not take up the compounds, and the drugs are inactive against the liver stages of Plasmodium berghei, a mouse malaria parasite. Our findings provide an explanation for the observed differences in activity of fosmidomycin and FR900098 against different Apicomplexa. These results have important implications for future screens aimed at finding new and safe molecular entities active against P. falciparum and related parasites. Our data provide further evidence that parasite-induced new permeability pathways may be exploited as routes for drug delivery.

Toxoplasma gondii is a zoonotic intracellular parasite, able to infect any warm-blooded animal via ingestion of infective stages, either contained in tissue cysts or oocysts released into the environment. While immune responses during infection are well-studied, there is still limited knowledge about the very early infection events in the gut tissue after infection via the oral route. Here we briefly discuss differences in host-specific responses following infection with oocyst-derived sporozoites vs. tissue cyst-derived bradyzoites. A focus is given to innate intestinal defense mechanisms and early immune cell events that precede T. gondii's dissemination in the host. We propose stem cell-derived intestinal organoids as a model to study early events of natural host-pathogen interaction. These offer several advantages such as live cell imaging and transcriptomic profiling of the earliest invasion processes. We additionally highlight the necessity of an appropriate large animal model reflecting human infection more closely than conventional infection models, to study the roles of dendritic cells and macrophages during early infection.

Toxoplasma gondii is an ubiquitous intracellular protozoan parasite. Mammals and birds are intermediate hosts and felid species are definitive hosts. In most human altered habitats the domestic cat is the predominant definitive host. Current knowledge of T. gondii infection in African ecosystems is limited. This study aimed to assess exposure to T. gondii in wild carnivores in the Serengeti ecosystem in East Africa. Carnivores can be infected by the consumption of tissue cysts when feeding on infected animals and by incidental ingestion of oocysts from environmental contamination. Incidental ingestion should occur regardless of a species' diet whereas the consumption of cysts should increase the chance of infection in carnivorous species. This predicts higher seropositivity in carnivorous than in insectivorous carnivores and lower seropositivity in juvenile carnivores with a long dependency on milk than in adults. We found high seropositivity in carnivorous species: 100% (15 of 15 samples) in adult African lions, 93% (38 of 41 samples) in adult spotted hyenas and one striped hyena sample was positive, whereas all four samples from the insectivorous bat-eared fox were negative. Juvenile hyenas (11 of 19 sera) had significantly lower seropositivity than adults (38 of 41 sera). Long-term monitoring of spotted hyenas revealed no significant difference in seropositivity between two periods (1988-1992 and 2000 to 2016). Identical results were produced in lion and hyena samples by a commercial multi-species ELISA (at serum dilution 1:10) and an in-house ELISA based on a recombinant T. gondii protein (at serum dilution 1:100), making the latter a useful alternative for small amounts of serum. We suggest that diet, age and lifetime range are factors determining seropositivity in carnivores in the Serengeti ecosystem and suggest that the role of small wild felids in the spread of T. gondii in the African ecosystem warrants investigation.

Rodents, in particular Mus musculus, have a long and invaluable history as models for human diseases in biomedical research, although their translational value has been challenged in a number of cases. We provide some examples in which rodents have been suboptimal as models for human biology and discuss confounders which influence experiments and may explain some of the misleading results. Infections of rodents with protozoan parasites are no exception in requiring close consideration upon model choice. We focus on the significant differences between inbred, outbred and wild animals, and the importance of factors such as microbiota, which are gaining attention as crucial variables in infection experiments. Frequently, mouse or rat models are chosen for convenience, e.g., availability in the institution rather than on an unbiased evaluation of whether they provide the answer to a given question. Apart from a general discussion on translational success or failure, we provide examples where infections with single-celled parasites in a chosen lab rodent gave contradictory or misleading results, and when possible discuss the reason for this. We present emerging alternatives to traditional rodent models, such as humanized mice and organoid primary cell cultures. So-called recombinant inbred strains such as the Collaborative Cross collection are also a potential solution for certain challenges. In addition, we emphasize the advantages of using wild rodents for certain immunological, ecological and/or behavioral questions. The experimental challenges (e.g. availability of species-specific reagents) that come with the use of such non-model systems are also discussed. Our intention is to foster critical judgment of both traditional and newly available translational rodent models for research on parasitic protozoa that can complement the existing mouse and rat models.

Toxoplasma gondii oocysts are highly infectious and may survive in the environment for years.