The genus Eimeria (Apicomplexa, Coccidia) provides a wide range of different species with different hosts to study common and variable features within the genus and its species. A common characteristic of all known Eimeria species is the oocyst, the infectious stage where its life cycle starts and ends. In our study, we utilized Eimeria nieschulzi as a model organism. This rat-specific parasite has complex oocyst morphology and can be transfected and even cultivated in vitro up to the oocyst stage. We wanted to elucidate how the known oocyst wall-forming proteins are preserved in this rodent Eimeria species compared to other Eimeria. In newly obtained genomics data, we were able to identify different gametocyte genes that are orthologous to already known gam genes involved in the oocyst wall formation of avian Eimeria species. These genes appeared putatively as single exon genes, but cDNA analysis showed alternative splicing events in the transcripts. The analysis of the translated sequence revealed different conserved motifs but also dissimilar regions in GAM proteins, as well as polymorphic regions. The occurrence of an underrepresented gam56 gene version suggests the existence of a second distinct E. nieschulzi genotype within the E. nieschulzi Landers isolate that we maintain.

Eimeria species are important veterinary coccidian parasites and are transmitted between hosts via oocysts. The infectious sporozoites are protected by the oocyst and sporocyst wall. Tyrosine-rich proteins are well-known components of the Eimeria oocyst wall. Recently, cysteine motif containing proteins (COWP family), as described in Toxoplasma gondii and Cryptosporidium spp., have also been characterized in Eimeria. Here, we identified a novel COWP-related protein, EnOWP13, and tracked it via transfection technology in Eimeria nieschulzi. The subsequent analysis suggests that the mCherry-tagged EnOWP13 localizes to the wall-forming bodies I and the outer wall. Immunohistochemical analysis confirmed the distribution of wall-forming bodies similar to avian Eimeria species and revealed that the wall-forming bodies I show peroxidase activity. The EnOWP13 amino acid composition and FITC-cadaverine-positive wall-forming bodies I suggest a participation of an enzyme with transglutaminase activity. This is the first description and characterization of this novel outer oocyst wall protein, which is also orthologous to other Eimeria species and Toxoplasma gondii, suggesting a new potential cross-linking mechanism of wall-forming proteins via isopeptide bonds.

Finding sustainable feed alternatives is an emerging topic in times of depletion of potential arable land and strict land use regulations. Black soldier fly (Hermetia illucens) larvae can be reared on almost all organic matters and may be used as a source for animal feed. However, the risk of disease transmission is high when animals are fed larvae or prepupae raised on waste materials that may contain potentially infectious pathogens. We qualitatively examined the effect of larval intestine extracts on the coccidian parasites Eimeria nieschulzi and Eimeria tenella and on eggs of the nematode Ascaris suum. Furthermore, we focused on the question of whether the persistent parasite stages (oocysts and eggs) would be digested, pass through living larvae, or attach to the larval surface. Neither living black soldier fly larvae nor black soldier fly larval intestine extracts had any effect on oocysts or eggs of the studied parasites. Thus, untreated H. illucens larvae as animal feed pose a risk of disease transmission to animals and humans, and a simple larval washing step is not sufficient for total removal of parasites. HIGHLIGHTS

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Coccidian parasites possess complex life cycles involving asexual proliferation followed by sexual development leading to the production of oocysts. Coccidian oocysts are persistent stages which are secreted by the feces and transmitted from host to host guaranteeing life cycle progression and disease transmission. The robust bilayered oocyst wall is formed from the contents of two organelles, the wall-forming bodies type I and II (WFBI, WFBII), located exclusively in the macrogametocyte. Eimeria nieschulzi has been used as a model parasite to study and follow gametocyte and oocyst development. In this study, the gametocyte and oocyst wall formation of E. nieschulzi was analyzed by electron microscopy and immuno-histology. A monoclonal antibody raised against the macrogametocytes of E. nieschulzi identified a tyrosine-rich glycoprotein (EnGAM82) located in WFBII. Correlative light and electron microscopy was used to examine the vesicle-specific localization and spatial distribution of GAM82-proteins during macrogametocyte maturation by this monoclonal antibody. In early and mid-stages, the GAM82-protein is ubiquitously distributed in WFBII. Few hours later, the protein is arranged in subvesicular structures. It was possible to show that the substructure of WFBII and the spatial distribution of GAM82-proteins probably represent pre-synthesized cross-linked materials prior to the inner oocyst wall formation. Dityrosine-cross-linked gametocyte proteins can also be confirmed and visualized by fluorescence microscopy (UV light, autofluorescence of WFBII). Electronic supplementary material The online version of this article (10.1007/s00436-020-06765-6) contains supplementary material, which is available to authorized users.