The Absence of Gut Microbiota Alters the Development of the Apicomplexan Parasite Eimeria tenella.

Coccidiosis is a widespread intestinal disease of poultry caused by a parasite of the genus Eimeria. Eimeria tenella, is one of the most virulent species that specifically colonizes the caeca, an organ which harbors a rich and complex microbiota. Our objective was to study the impact of the intestinal microbiota on parasite infection and development using an original model of germ-free broilers. We observed that germ-free chickens presented significantly much lower load of oocysts in caecal contents than conventional chickens. This decrease in parasite load was measurable in caecal tissue by RT-qPCR at early time points. Histological analysis revealed the presence of much less first (day 2pi) and second generation schizonts (day 3.5pi) in germ-free chickens than conventional chickens. Indeed, at day 3.5pi, second generation schizonts were respectively immature only in germ-free chickens suggesting a lengthening of the asexual phase of the parasite in the absence of microbiota. Accordingly to the consequence of this lengthening, a delay in specific gamete gene expressions, and a reduction of gamete detection by histological analysis in caeca of germ-free chickens were observed. These differences in parasite load might result from an initial reduction of the excystation efficiency of the parasite in the gut of germ-free chickens. However, as bile salts involved in the excystation step led to an even higher excystation efficiency in germ-free compared to conventional chickens, this result could not explain the difference in parasite load. Interestingly, when we shunted the excystation step in vivo by infecting chickens with sporozoites using the cloacal route of inoculation, parasite invasion was similar in germ-free and in conventional chickens but still resulted in significantly lower parasite load in germ-free chickens at day 7pi. Overall, these data highlighted that the absence of intestinal microbiota alters E. tenella replication. Strategies to modulate the microbiota and/or its metabolites could therefore be an alternative approach to limit the negative impact of coccidiosis in poultry.

Chicken endothelial cells are highly responsive to viral innate immune stimuli and are susceptible to infections with various avian pathogens.

It is well established that the endothelium plays a prominent role in the pathogenesis of various infectious diseases in mammals. However, little is known about the role of endothelial cells (EC) as targets for avian pathogens and their contribution to the pathogenesis of infectious diseases in galliform birds. First, we explored the innate immune response of primary chicken aortic endothelial cells (pchAEC), obtained from 18-day-old embryos, to stimulation with pathogen-associated molecular patterns or recombinant chicken interferons (type I, II and III IFNs). In spite of the abundant expression of a number of innate immune receptors, marked cytokine responses to stimulation with pathogen-associated molecular patterns were only seen in pchAEC treated with the TLR3 agonist polyI:C (pI:C) and the MDA5 agonist liposome-complexed polyI:C (L-pI:C), as was assessed by quantitative PCR and luciferase-based IFN-I/NFκB reporter assays. Treatments of pchAEC with IFN-α, IFN-γ and IFN-λ resulted in STAT1-phosphorylation/activation, as was revealed by immunoblotting. Next, we demonstrated that pchAEC are susceptible to infection with a variety of poultry pathogens, including Marek's disease virus (MDV), infectious bursal disease virus (IBDV), avian pathogenic Escherichia coli (APEC) and Eimeria tenella. Our data highlight that chicken EC are potential targets for viral, bacterial and protozoan pathogens in gallinaceous poultry and may partake in the inflammatory and antimicrobial response. The pchAEC infection model used herein will allow further studies interrogating avian pathogen interactions with vascular EC. RESEARCH HIGHLIGHTS Use of a well-defined primary chicken aortic endothelial cell (pchAEC) culture model for studying avian host-pathogen interactions. pchAEC are responsive to innate immune stimulation with viral pathogen-associated molecular patterns and chicken type I, II and III interferons. pchAEC are susceptible to infections with economically important poultry pathogens, including MDV, IBDV, APEC and Eimeria tenella.

Establishment of an in vitro chicken epithelial cell line model to investigate Eimeria tenella gamete development.

BACKGROUND: Eimeria tenella infection leads to acute intestinal disorders responsible for important economic losses in poultry farming worldwide. The life-cycle of E. tenella is monoxenous with the chicken as the exclusive host; infection occurs in caecal epithelial cells. However, in vitro, the complete life-cycle of the parasite has only been propagated successfully in primary chicken kidney cells, which comprise undefined mixed cell populations; no cell line model has been able to consistently support the development of the sexual stages of the parasite. We therefore sought to develop a new model to study E. tenella gametogony in vitro using a recently characterised chicken cell line (CLEC-213) exhibiting an epithelial cell phenotype. METHODS: CLEC-213 were infected with sporozoites from a precocious strain or with second generation merozoites (merozoites II) from wild type strains. Sexual stages of the parasite were determined both at the gene and protein levels. RESULTS: To our knowledge, we show for the first time in CLEC-213, that sporozoites from a precocious strain of E. tenella were able to develop to gametes, as verified by measuring gene expression and by using antibodies to a microgamete-specific protein (EtFOA1: flagellar outer arm protein 1) and a macrogamete-specific protein (EtGAM-56), but oocysts were not observed. However, both gametes and oocysts were observed when cells were infected with merozoites II from wild type strains, demonstrating that completion of the final steps of the parasite cycle is possible in CLEC-213 cells. CONCLUSION: The epithelial cell line CLEC-213 constitutes a useful avian tool for studying Eimeria epithelial cell interactions and the effect of drugs on E. tenella invasion, merogony and gametogony.

Discovery of a tyrosine-rich sporocyst wall protein in Eimeria tenella.

BACKGROUND: Eimeria is an important genus of apicomplexan parasites. A defining feature of these parasites is the oocyst, which is transmitted into the environment via the faeces of definitive hosts. The oocyst wall contains cross-linked, tyrosine-rich proteins and protects eight infectious sporozoites, housed in pairs within a second walled structure, the sporocyst. The biochemical basis for sporocyst wall formation is not known. FINDINGS: Here, we report the discovery of a novel tyrosine-rich protein, EtSWP1, in Eimeria tenella. Like the tyrosine-rich proteins of the oocyst wall, EtSWP1 is an intrinsically disordered protein with the tyrosine residues concentrated in a specific region of the protein, located immediately following the region of intrinsic disorder. We engineered E. tenella to express mCherry-tagged EtSWP1 and showed that the tagged protein localises specifically to sporocyst walls, indicating that the biochemistry of sporocyst wall assembly is analagous to that of oocyst walls. CONCLUSIONS: Tyrosine-rich proteins are known to be key components of the oocyst wall and we now demonstrate, using gene and protein analyses combined with genetic manipulation, that a novel tyrosine-rich protein is specific for the sporocyst wall. This finding is important because it shows that the biochemistry of these two distinct walls is similar and, hence, brings targeted disruption of sporulation and, therefore, potential neutralisation of oocysts in the environment, a step closer.

Synthesis and evaluation of the anticoccidial activity of trifluoropyrido[1,2-a]pyrimidin-2-one derivatives.

Screening of our chemical library to discover new molecules exhibiting in vitro activity against the invasion of host cells by Eimeria tenella revealed a lead compound with an IC50 of 15µM. Structure-activity relationship studies were conducted with 34 newly synthesized compounds to identify more active molecules and enhance in vitro activity against the parasite. Four compounds were more effective in inhibiting MDBK cell invasion in vitro than the lead compound.

Reduced parasite motility and micronemal protein secretion by a p38 MAPK inhibitor leads to a severe impairment of cell invasion by the apicomplexan parasite Eimeria tenella.

E. tenella infection is associated with a severe intestinal disease leading to high economic losses in poultry industry. Mitogen activated protein kinases (MAPKs) are implicated in early response to infection and are divided in three pathways: p38, extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK). Our objective was to determine the importance of these kinases on cell invasion by E. tenella. We evaluated the effect of specific inhibitors (ERK: PD98059, JNKII: SP600125, p38 MAPK: SB203580) on the invasion of epithelial cells. Incubation of SP600125 and SB203580 with epithelial cells and parasites significantly inhibited cell invasion with the highest degree of inhibition (90%) for SB203580. Silencing of the host p38α MAPK expression by siRNA led to only 20% decrease in cell invasion. In addition, when mammalian epithelial cells were pre-treated with SB203580, and washed prior infection, a 30% decrease in cell invasion was observed. This decrease was overcome when a p38 MAPK activator, anisomycin was added during infection. This suggests an active but limited role of the host p38 MAPK in this process. We next determined whether SB203580 has a direct effect on the parasite. Indeed, parasite motility and secretion of micronemal proteins (EtMIC1, 2, 3 and 5) that are involved in cell invasion were both decreased in the presence of the inhibitor. After chasing the inhibitor, parasite motility and secretion of micronemal proteins were restored and subsequently cell invasion. SB203580 inhibits cell invasion by acting partly on the host cell and mainly on the parasite.

Aminopeptidase N1 (EtAPN1), an M1 metalloprotease of the apicomplexan parasite Eimeria tenella, participates in parasite development.

Aminopeptidases N are metalloproteases of the M1 family that have been reported in numerous apicomplexan parasites, including Plasmodium, Toxoplasma, Cryptosporidium, and Eimeria. While investigating the potency of aminopeptidases as therapeutic targets against coccidiosis, one of the most important avian diseases caused by the genus Eimeria, we identified and characterized Eimeria tenella aminopeptidase N1 (EtAPN1). Its inhibition by bestatin and amastatin, as well as its reactivation by divalent ions, is typical of zinc-dependent metalloproteases. EtAPN1 shared a similar sequence, three-dimensional structure, and substrate specificity and similar kinetic parameters with A-M1 from Plasmodium falciparum (PfA-M1), a validated target in the treatment of malaria. EtAPN1 is synthesized as a 120-kDa precursor and cleaved into 96-, 68-, and 38-kDa forms during sporulation. Further, immunolocalization assays revealed that, similar to PfA-M1, EtAPN1 is present during the intracellular life cycle stages in both the parasite cytoplasm and the parasite nucleus. The present results support the hypothesis of a conserved role between the two aminopeptidases, and we suggest that EtAPN1 might be a valuable target for anticoccidiosis drugs.

Discovery of compounds blocking the proliferation of Toxoplasma gondii and Plasmodium falciparum in a chemical space based on piperidinyl-benzimidazolone analogs.

A piperidinyl-benzimidazolone scaffold has been found in the structure of different inhibitors of membrane glycerolipid metabolism, acting on enzymes manipulating diacylglycerol and phosphatidic acid. Screening a focus library of piperidinyl-benzimidazolone analogs might therefore identify compounds acting against infectious parasites. We first evaluated the in vitro effects of (S)-2-(dibenzylamino)-3-phenylpropyl 4-(1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)piperidine-1-carboxylate (compound 1) on Toxoplasma gondii and Plasmodium falciparum. In T. gondii, motility and apical complex integrity appeared to be unaffected, whereas cell division was inhibited at compound 1 concentrations in the micromolar range. In P. falciparum, the proliferation of erythrocytic stages was inhibited, without any delayed death phenotype. We then explored a library of 250 analogs in two steps. We selected 114 compounds with a 50% inhibitory concentration (IC50) cutoff of 2 µM for at least one species and determined in vitro selectivity indexes (SI) based on toxicity against K-562 human cells. We identified compounds with high gains in the IC50 (in the 100 nM range) and SI (up to 1,000 to 2,000) values. Isobole analyses of two of the most active compounds against P. falciparum indicated that their interactions with artemisinin were additive. Here, we propose the use of structure-activity relationship (SAR) models, which will be useful for designing probes to identify the target compound(s) and optimizations for monotherapy or combined-therapy strategies.

Eimeripain, a cathepsin B-like cysteine protease, expressed throughout sporulation of the apicomplexan parasite Eimeria tenella.

The invasion and replication of Eimeria tenella in the chicken intestine is responsible for avian coccidiosis, a disease that has major economic impacts on poultry industries worldwide. E. tenella is transmitted to naïve animals via shed unsporulated oocysts that need contact with air and humidity to form the infectious sporulated oocysts, which contain the first invasive form of the parasite, the sporozoite. Cysteine proteases (CPs) are major virulence factors expressed by protozoa. In this study, we show that E. tenella expresses five transcriptionally regulated genes encoding one cathepsin L, one cathepsin B and three cathepsin Cs. Biot-LC-LVG-CHN2, a cystatin derived probe, tagged eight polypeptides in unsporulated oocysts but only one in sporulated oocysts. CP-dependant activities were found against the fluorescent substrates, Z-FR-AMC and Z-LR-AMC, throughout the sporulation process. These activities corresponded to a cathepsin B-like enzyme since they were inhibited by CA-074, a specific cathepsin B inhibitor. A 3D model of the catalytic domain of the cathepsin B-like protease, based on its sequence homology with human cathepsin B, further confirmed its classification as a papain-like protease with similar characteristics to toxopain-1 from the related apicomplexan parasite, Toxoplasma gondii; we have, therefore, named the E. tenella cathepsin B, eimeripain. Following stable transfection of E. tenella sporozoites with a plasmid allowing the expression of eimeripain fused to the fluorescent protein mCherry, we demonstrated that eimeripain is detected throughout sporulation and has a punctate distribution in the bodies of extra- and intracellular parasites. Furthermore, CA-074 Me, the membrane-permeable derivative of CA-074, impairs invasion of epithelial MDBK cells by E. tenella sporozoites. This study represents the first characterization of CPs expressed by a parasite from the Eimeria genus. Moreover, it emphasizes the role of CPs in transmission and dissemination of exogenous stages of apicomplexan parasites.

Development of a high-sensitivity nested PCR assay for the detection of Clostridium piliforme in clinical samples.

Tyzzer's disease has been reported in numerous animal species, including rodents and lagomorphs such as the rabbit. Because the organism cannot be cultivated on artificial media the diagnosis of the aetiological agent Clostridium piliforme is rather difficult and is based on the identification of typical gross lesions and histological demonstration of the characteristic intracellular bacteria at the periphery of the necrotic foci. To address the limitations of C. piliforme detection a nested polymerase chain reaction (nPCR) assay has been developed with increased specificity with regard to the phylogenetically close bacterial strains (based on 16S RNA sequence analogies). The sensitivity of this nPCR was also demonstrated using clinical samples.