What Do We Know about Surface Proteins of Chicken Parasites Eimeria?

Poultry is the first source of animal protein for human consumption. In a changing world, this sector is facing new challenges, such as a projected increase in demand, higher standards of food quality and safety, and reduction of environmental impact. Chicken coccidiosis is a highly widespread enteric disease caused by Eimeria spp. which causes significant economic losses to the poultry industry worldwide; however, the impact on family poultry holders or backyard production-which plays a key role in food security in small communities and involves mainly rural women-has been little explored. Coccidiosis disease is controlled by good husbandry measures, chemoprophylaxis, and/or live vaccination. The first live vaccines against chicken coccidiosis were developed in the 1950s; however, after more than seven decades, none has reached the market. Current limitations on their use have led to research in next-generation vaccines based on recombinant or live-vectored vaccines. Next-generation vaccines are required to control this complex parasitic disease, and for this purpose, protective antigens need to be identified. In this review, we have scrutinised surface proteins identified so far in Eimeria spp. affecting chickens. Most of these surface proteins are anchored to the parasite membrane by a glycosylphosphatidylinositol (GPI) molecule. The biosynthesis of GPIs, as well as the role of currently identified surface proteins and interest as vaccine candidates has been summarised. The potential role of surface proteins in drug resistance and immune escape and how these could limit the efficacy of control strategies was also discussed.

Refractile bodies of Eimeria tenella are proteinaceous membrane-less organelles that undergo dynamic changes during infection.

Introduction: Refractile bodies (RB) are large membrane-less organelles (MLO) of unknown function found as a prominent mismatched pair within the sporozoite stages of all species of Eimeria, parasitic coccidian protozoa. Methods: High resolution imaging methods including time-lapse live confocal microscopy and serial block face-scanning electron microscopy (SBF-SEM) were used to investigate the morphology of RB and other intracellular organelles before and after sporozoite invasion of host cells. Results: Live cell imaging of MDBK cells infected with E. tenella sporozoites confirmed previous reports that RB reduce from two to one post-infection and showed that reduction in RB number occurs via merger of the anterior RB with the posterior RB, a process that lasts 20-40 seconds and takes place between 2- and 5-hours post-infection. Ultrastructural studies using SBF-SEM on whole individual sporozoites, both pre- and post-host cell invasion, confirmed the live cell imaging observations and showed also that changes to the overall sporozoite cell shape accompanied RB merger. Furthermore, the single RB post-merger was found to be larger in volume than the two RB pre-merger. Actin inhibitors were used to investigate a potential role for actin in RB merger, Cytochalasin D significantly inhibited both RB merger and the accompanying changes in sporozoite cell shape. Discussion: MLOs in eukaryotic organisms are characterised by their lack of a membrane and ability to undergo liquid-liquid phase separation (LLPS) and fusion, usually in an actin-mediated fashion. Based on the changes in sporozoite cell shape observed at the time of RB merger together with a potential role for actin in this process, we propose that RB are classed as an MLO and recognised as one of the largest MLOs so far characterised.

Thyme, Oregano, and Garlic Essential Oils and Their Main Active Compounds Influence Eimeria tenella Intracellular Development.

Coccidiosis poses a significant challenge in poultry production and is typically managed with ionophores and chemical anticoccidials. However, the emergence of drug resistance and limitations on their use have encouraged the exploration of alternative solutions, including botanical compounds and improvements in in vitro screening methods. Prior research focused only on the impact of these alternatives on Eimeria invasion, with intracellular development in cell cultures receiving limited attention. This study assessed the impact of thyme (Thymus vulgaris), oregano (Origanum vulgare), and garlic (Allium sativum) essential oils, as well as their bioactive compounds, on the initial phase of schizogony in Madin-Darby bovine kidney cells, comparing their effectiveness to two commercially used anticoccidial drugs. Using image analysis and quantitative PCR, the study confirmed the efficacy of commercial anticoccidials in reducing invasion and schizont formation, and it found that essential oils were equally effective. Notably, thymol and carvacrol exhibited mild inhibition of intracellular replication of the parasite but significantly reduced schizont numbers, implying a potential reduction in pathogenicity. In conclusion, this research highlights the promise of essential oils and their bioactive components as viable alternatives to traditional anticoccidial drugs for mitigating coccidiosis in poultry, particularly by disrupting the intracellular development of the parasites.

In Vitro Antioxidant, Antimicrobial, Anticoccidial, and Anti-Inflammatory Study of Essential Oils of Oregano, Thyme, and Sage from Epirus, Greece.

Origanum vulgare subsp. hirtum, Thymus vulgaris, and Salvia fructicosa are aromatic plants commonly found in Mediterranean countries and are traditionally used in Greece as a remedy for humans, since they are well known as potent antibacterial, antioxidant, and anti-inflammatory agents. Essential oils (EOs) derived from plants cultivated in the mountainous region of Epirus, Greece, were investigated for their inhibitory activity against key microorganisms with relevance to avian health, while also assessing their antioxidant and anti-inflammatory activity. The total phenolic content (TPC) of the EOs was estimated according to the Folin−Ciocalteu method, while the antioxidant capacity was tested through the EOs' ability to scavenge free radicals by means of the DPPH, ABTS, and FRAP assays. Antibacterial and anti-inflammatory effects were examined by the agar disc diffusion method and the lipoxygenase (LOX) inhibition test, respectively. Furthermore, the EOs' ability to inhibit the invasion of sporozoites of Eimeria tenella (Wisconsin strain) along with any toxic effects were assayed in Madin−Darby bovine kidney (MDBK) cells. The antioxidant activity of the EOs was observed in descending order: oregano > thyme > sage. The antimicrobial effects of thyme and oregano were equivalent and higher than that of sage, while the anti-inflammatory effect of thyme was higher compared to both sage and oregano. The intracellular invasion of sporozoites was evaluated by the detection of E. tenella DNA by qPCR from cell monolayers harvested at 2 and 24 h post-infection. Parasite invasion was inhibited by the addition of oregano essential oil at the concentration of 100 µg/mL by 83% or 93% after 2 or 24 h, respectively, and was higher compared to the addition of thyme and sage, which had similar effects, but at a less intensive level. The cytotoxic assessment of all three essential oils revealed that they had no effect on MDBK cells compared to dimethyl sulfoxide (DMSO), used as the control substance. The supplementation of oregano, thyme, and sage essential oils had a potent antioxidant, anti-inflammatory, antimicrobial, and anticoccidial in vitro effect that is comparable to synthetic substances or approved drugs, justifying the need for further evaluation by in vivo studies in broilers reared in the absence of antimicrobial and anticoccidial drugs or synthetic antioxidant and/or anti-inflammatory compounds.

Cellular electron tomography of the apical complex in the apicomplexan parasite Eimeria tenella shows a highly organised gateway for regulated secretion.

The apical complex of apicomplexan parasites is essential for host cell invasion and intracellular survival and as the site of regulated exocytosis from specialised secretory organelles called rhoptries and micronemes. Despite its importance, there are few data on the three-dimensional organisation and quantification of these organelles within the apical complex or how they are trafficked to this specialised region of plasma membrane for exocytosis. In coccidian apicomplexans there is an additional tubulin-containing hollow barrel structure, the conoid, which provides a structural gateway for this specialised apical secretion. Using a combination of cellular electron tomography and serial block face-scanning electron microscopy (SBF-SEM) we have reconstructed the entire apical end of Eimeria tenella sporozoites; we report a detailed dissection of the three- dimensional organisation of the conoid and show there is high curvature of the tubulin-containing fibres that might be linked to the unusual comma-shaped arrangement of protofilaments. We quantified the number and location of rhoptries and micronemes within cells and show a highly organised gateway for trafficking and docking of rhoptries, micronemes and microtubule-associated vesicles within the conoid around a set of intra-conoidal microtubules. Finally, we provide ultrastructural evidence for fusion of rhoptries directly through the parasite plasma membrane early in infection and the presence of a pore in the parasitophorous vacuole membrane, providing a structural explanation for how rhoptry proteins may be trafficked between the parasite and the host cytoplasm.

A Novel Whole Yeast-Based Subunit Oral Vaccine Against Eimeria tenella in Chickens.

Cheap, easy-to-produce oral vaccines are needed for control of coccidiosis in chickens to reduce the impact of this disease on welfare and economic performance. Saccharomyces cerevisiae yeast expressing three Eimeria tenella antigens were developed and delivered as heat-killed, freeze-dried whole yeast oral vaccines to chickens in four separate studies. After vaccination, E. tenella replication was reduced following low dose challenge (250 oocysts) in Hy-Line Brown layer chickens (p<0.01). Similarly, caecal lesion score was reduced in Hy-Line Brown layer chickens vaccinated using a mixture of S. cerevisiae expressing EtAMA1, EtIMP1 and EtMIC3 following pathogenic-level challenge (4,000 E. tenella oocysts; p<0.01). Mean body weight gain post-challenge with 15,000 E. tenella oocysts was significantly increased in vaccinated Cobb500 broiler chickens compared to mock-vaccinated controls (p<0.01). Thus, inactivated recombinant yeast vaccines offer cost-effective and scalable opportunities for control of coccidiosis, with relevance to broiler production and chickens reared in low-and middle-income countries (LMICs).

Controlling the causative agents of coccidiosis in domestic chickens; an eye on the past and considerations for the future.

Coccidiosis is a potentially severe enteritis caused by species of obligate intracellular parasites of the genus Eimeria. These parasites cause significant economic losses to the poultry industry, predominantly due to compromised efficiency of production as well as the cost of control. These losses were recently estimated to cost chicken producers approximately £10.4 billion worldwide annually. High levels of Eimeria infection cause clinical coccidiosis which is a significant threat to poultry welfare, and a pre-disposing contributory factor for necrotic enteritis. Control of Eimeria parasites and coccidiosis is therefore an important endeavour; multiple approaches have been developed and these are often deployed together. This review summarises current trends in strategies for control of Eimeria, focusing on three main areas: good husbandry, chemoprophylaxis and vaccination. There is currently no "perfect solution" and there are advantages and limitations to all existing methods. Therefore, the aim of this review is to present current control strategies and suggest how these may develop in the future.

Do All Coccidia Follow the Same Trafficking Rules?

The Coccidia are a subclass of the Apicomplexa and include several genera of protozoan parasites that cause important diseases in humans and animals, with Toxoplasma gondii becoming the 'model organism' for research into the coccidian molecular and cellular processes. The amenability to the cultivation of T. gondii tachyzoites and the wide availability of molecular tools for this parasite have revealed many mechanisms related to their cellular trafficking and roles of parasite secretory organelles, which are critical in parasite-host interaction. Nevertheless, the extrapolation of the T. gondii mechanisms described in tachyzoites to other coccidian parasites should be done carefully. In this review, we considered published data from Eimeria parasites, a coccidian genus comprising thousands of species whose infections have important consequences in livestock and poultry. These studies suggest that the Coccidia possess both shared and diversified mechanisms of protein trafficking and secretion potentially linked to their lifecycles. Whereas trafficking and secretion appear to be well conversed prior to and during host-cell invasion, important differences emerge once endogenous development commences. Therefore, further studies to validate the mechanisms described in T. gondii tachyzoites should be performed across a broader range of coccidians (including T. gondii sporozoites). In addition, further genus-specific research regarding important disease-causing Coccidia is needed to unveil the individual molecular mechanisms of pathogenesis related to their specific lifecycles and hosts.

Spotlight on avian pathology: Eimeria and the disease coccidiosis.

Coccidiosis, caused by Eimeria species parasites, remains a major threat to poultry production, undermining economic performance and compromising welfare. The recent characterization of three new Eimeria species that infect chickens has highlighted that many gaps remain in our knowledge of the biology and epidemiology of these parasites. Concerns about the use of anticoccidial drugs, widespread parasite drug resistance, the need for vaccines that can be used across broiler as well as layer and breeder sectors, and consumer preferences for "clean" farming, all point to the need for novel control strategies. New research tools including vaccine delivery vectors, high throughput sequencing, parasite transgenesis and sensitive molecular assays that can accurately assess parasite development in vitro and in vivo are all proving helpful in the ongoing quest for improved cost-effective, scalable strategies for future control of coccidiosis.

Impact of Eimeria tenella Oocyst Dose on Parasite Replication, Lesion Score and Cytokine Transcription in the Caeca in Three Breeds of Commercial Layer Chickens.

Eimeria species parasites infect the gastrointestinal tract of chickens, causing disease and impacting on production. The poultry industry relies on anticoccidial drugs and live vaccines to control Eimeria and there is a need for novel, scalable alternatives. Understanding the outcomes of experimental infection in commercial chickens is valuable for assessment of novel interventions. We examined the impact of different infectious doses of Eimeria tenella (one low dose, three high doses) in three commercial layer chicken lines, evaluating lesion score, parasite replication and cytokine response in the caeca. Groups of eight to ten chickens were housed together and infected with 250, 4,000, 8,000 or 12,000 sporulated oocysts at 21 days of age. Five days post-infection caeca were assessed for lesions and to quantify parasite replication by qPCR and cytokine transcription by RT-qPCR. Comparison of the three high doses revealed no significant variation between them in observed lesions or parasite replication with all being significantly higher than the low dose infection. Transcription of IFN-γ and IL-10 increased in all infected chickens relative to unchallenged controls, with no significant differences associated with dose magnitude (p > 0.05). No significant differences were detected in lesion score, parasite replication or caecal cytokine expression between the three lines of chickens. We therefore propose 4,000 E. tenella oocysts is a sufficient dose to reliably induce lesions in commercial layer chickens, and that estimates of parasite replication can be derived by qPCR from these same birds. However, more accurate quantification of Eimeria replication requires a separate low dose challenge group. Optimisation of challenge dose in an appropriate chicken line is essential to maximize the value of in vivo efficacy studies. For coccidiosis, this approach can reduce the numbers of chickens required for statistically significant studies and reduce experimental severity.

Next-Generation Technologies and Systems Biology for the Design of Novel Vaccines Against Apicomplexan Parasites.

Parasites of the phylum Apicomplexa are the causative agents of important diseases such as malaria, toxoplasmosis or cryptosporidiosis in humans, and babesiosis and coccidiosis in animals. Whereas the first human recombinant vaccine against malaria has been approved and recently recommended for wide administration by the WHO, most other zoonotic parasitic diseases lack of appropriate immunoprophylaxis. Sequencing technologies, bioinformatics, and statistics, have opened the "omics" era into apicomplexan parasites, which has led to the development of systems biology, a recent field that can significantly contribute to more rational design for new vaccines. The discovery of novel antigens by classical approaches is slow and limited to very few antigens identified and analyzed by each study. High throughput approaches based on the expansion of the "omics", mainly genomics and transcriptomics have facilitated the functional annotation of the genome for many of these parasites, improving significantly the understanding of the parasite biology, interactions with the host, as well as virulence and host immune response. Developments in genetic manipulation in apicomplexan parasites have also contributed to the discovery of new potential vaccine targets. The present minireview does a comprehensive summary of advances in "omics", CRISPR/Cas9 technologies, and in systems biology approaches applied to apicomplexan parasites of economic and zoonotic importance, highlighting their potential of the holistic view in vaccine development.

The Growth of Eimeria tenella: Characterization and Application of Quantitative Methods to Assess Sporozoite Invasion and Endogenous Development in Cell Culture.

In vitro development of the complete life cycle of Eimeria species has been achieved in primary cultures of avian epithelial cells with low efficiency. The use of immortalized cell lines simplifies procedures but only allows partial development through one round of parasite invasion and intracellular replication. We have assessed the suitability of Madin-Darby Bovine Kidney (MDBK) cells to support qualitative and quantitative studies on sporozoite invasion and intracellular development of Eimeria tenella. Analysis of parasite ultrastructure by transmission electron microscopy and serial block face-scanning electron microscopy proved the suitability of the system to generate good quality schizonts and first-generation merozoites. Parasite protein expression profiles elucidated by mass spectrometry corroborated previous findings occurring during the development of the parasite such as the presence of alternative types of surface antigen at different stages and increased abundance of proteins from secretory organelles during invasion and endogenous development. Quantitative PCR (qPCR) allowed the tracking of development by detecting DNA division, whereas reverse transcription qPCR of sporozoite- and merozoite-specific genes could detect early changes before cell division and after merozoite formation, respectively. These results correlated with the analysis of development using ImageJ semi-automated image analysis of fluorescent parasites, demonstrating the suitability and reproducibility of the MDBK culture system. This systems also allowed the evaluation of the effects on invasion and development when sporozoites were pre-incubated with anticoccidial drugs, showing similar effects to those reported before. We have described through this study a series of methods and assays for the further application of this in vitro culture model to more complex studies of Eimeria including basic research on parasite cell biology and host-parasite interactions and for screening anticoccidial drugs.

In vitro Anticoccidial Study of Oregano and Garlic Essential Oils and Effects on Growth Performance, Fecal Oocyst Output, and Intestinal Microbiota in vivo.

This study investigated the in vitro effects of Greek oregano and garlic essential oils on inhibition of Eimeria parasites and their in vivo effects on production performance, intestinal bacteria counts, and oocyst output. An inhibition assay was performed in vitro using Eimeria tenella Wisconsin strain sporozoites and Madin-Darby bovine kidney (MDBK) cells. Intracellular sporozoite invasion was quantified by detection of E. tenella DNA using qPCR from cell monolayers harvested at 2 and 24 h post-infection. Parasite invasion was inhibited by the oregano essential oil at the concentration of 100 µg/ml by 83 or 93% after 2 or 24 h, respectively. Garlic essential oil reached a maximum inhibition of 70% after 24 h with the 50 µg/ml concentration. Normal morphology was observed in MDBK cells exposed to concentrations of 100 µl/ml of garlic or oregano for over 24 h. In the in vivo trial, 180 male broiler chicks (45.3 ± 0.7 g) were allocated into two treatments (6 pens of 15 chicks per treatment). Control treatment was fed commercial diets without antibiotics or anticoccidials. The ORE-GAR treatment was fed the same control diets, further supplemented with a premix (1 g/kg feed) containing the oregano (50 g/kg premix) and garlic (5 g/kg premix) essential oils. At day 37, all birds were slaughtered under commercial conditions, and intestinal samples were collected. ORE-GAR treatment had improved final body weight (1833.9 vs. 1.685.9 g; p < 0.01), improved feed conversion ratio (1.489 vs. 1.569; p < 0.01), and reduced fecal oocyst excretion (day 28: 3.672 vs. 3.989 log oocysts/g, p < 0.01; day 37: 3.475 vs. 4.007 log oocysts/g, p < 0.001). In the caecal digesta, ORE-GAR treatment had lower total anaerobe counts (8.216 vs. 8.824 CFU/g; p < 0.01), whereas in the jejunum digesta the ORE-GAR treatment had higher counts of E. coli (5.030 vs. 3.530 CFU/g; p = 0.01) and Enterobacteriaceae (5.341 vs. 3.829 CFU/g; p < 0.01), and lower counts of Clostridium perfringens (2.555 vs. 2.882 CFU/g; p < 0.01). In conclusion, the combined supplementation of oregano and garlic essential oils had a potent anticoccidial effect in vitro and a growth-promoting effect in broilers reared in the absence of anticoccidial drugs.

Vaccination with transgenic Eimeria tenella expressing Eimeria maxima AMA1 and IMP1 confers partial protection against high-level E. maxima challenge in a broiler model of coccidiosis.

BACKGROUND: Poultry coccidiosis is a parasitic enteric disease with a highly negative impact on chicken production. In-feed chemoprophylaxis remains the primary method of control, but the increasing ineffectiveness of anticoccidial drugs, and potential future restrictions on their use has encouraged the use of commercial live vaccines. Availability of such formulations is constrained by their production, which relies on the use of live chickens. Several experimental approaches have been taken to explore ways to reduce the complexity and cost of current anticoccidial vaccines including the use of live vectors expressing relevant Eimeria proteins. We and others have shown that vaccination with transgenic Eimeria tenella parasites expressing Eimeria maxima Apical Membrane Antigen-1 or Immune Mapped Protein-1 (EmAMA1 and EmIMP1) partially reduces parasite replication after challenge with a low dose of E. maxima oocysts. In the present study, we have reassessed the efficacy of these experimental vaccines using commercial birds reared at high stocking densities and challenged with both low and high doses of E. maxima to evaluate how well they protect chickens against the negative impacts of disease on production parameters. METHODS: Populations of E. tenella parasites expressing EmAMA1 and EmIMP1 were obtained by nucleofection and propagated in chickens. Cobb500 broilers were immunised with increasing doses of transgenic oocysts and challenged two weeks later with E. maxima to quantify the effect of vaccination on parasite replication, local IFN-γ and IL-10 responses (300 oocysts), as well as impacts on intestinal lesions and body weight gain (10,000 oocysts). RESULTS: Vaccination of chickens with E. tenella expressing EmAMA1, or admixtures of E. tenella expressing EmAMA1 or EmIMP1, was safe and induced partial protection against challenge as measured by E. maxima replication and severity of pathology. Higher levels of protection were observed when both antigens were delivered and was associated with a partial modification of local immune responses against E. maxima, which we hypothesise resulted in more rapid immune recognition of the challenge parasites. CONCLUSIONS: This study offers prospects for future development of multivalent anticoccidial vaccines for commercial chickens. Efforts should now be focused on the discovery of additional antigens for incorporation into such vaccines.

Life cycle stages, specific organelles and invasion mechanisms of Eimeria species.

Apicomplexans, including species of Eimeria, pose a real threat to the health and wellbeing of animals and humans. Eimeria parasites do not infect humans but cause an important economic impact on livestock, in particular on the poultry industry. Despite its high prevalence and financial costs, little is known about the cell biology of these 'cosmopolitan' parasites found all over the world. In this review, we discuss different aspects of the life cycle and stages of Eimeria species, focusing on cellular structures and organelles typical of the coccidian family as well as genus-specific features, complementing some 'unknowns' with what is described in the closely related coccidian Toxoplasma gondii.

Laboratory Growth and Genetic Manipulation of Eimeria tenella.

Eimeria is a genus of apicomplexan parasites that contains a large number of species, most of which are absolutely host-specific. Seven species have been recognized to infect chickens. Infection of susceptible chickens results in an intestinal disease called coccidiosis, characterized by mucoid or hemorrhagic enteritis, which is associated with impaired feed conversion or mortality in severe cases. Intensive farming practices have increased the significance of coccidiosis since parasite transmission is favored by high-density housing of large numbers of susceptible chickens. Routine chemoprophylaxis and/or vaccination with live parasite vaccines provides effective control of Eimeria, although the emergence of drug resistance and the relative cost and production capacity of current vaccine lines can prove limiting. As pressure to reduce drug use in livestock production intensifies, novel vaccination strategies are needed. Development of effective protocols supporting genetic complementation of Eimeria species has until recently been hampered by their inability to replicate efficiently in vitro. Now, the availability of such protocols has raised the prospect of generating transgenic parasite lines that function as vaccine vectors to express and deliver heterologous antigens. For example, this technology has the potential to streamline the production of live anticoccidial vaccines through the generation of parasite lines that co-express immunoprotective antigens derived from multiple Eimeria species. In this paper we describe detailed protocols for genetic manipulation, laboratory growth, and in vivo propagation of Eimeria tenella parasites, which will encourage future work from other researchers to expand biological understanding of Eimeria through reverse genetics. © 2019 by John Wiley & Sons, Inc.

Development of cross-protective Eimeria-vectored vaccines based on apical membrane antigens.

Recently, the availability of protocols supporting genetic complementation of Eimeria has raised the prospect of generating transgenic parasite lines which can function as vaccine vectors, expressing and delivering heterologous proteins. Complementation with sequences encoding immunoprotective antigens from other Eimeria spp. offers an opportunity to reduce the complexity of species/strains in anticoccidial vaccines. Herein, we characterise and evaluate EtAMA1 and EtAMA2, two members of the apical membrane antigen (AMA) family of parasite surface proteins from Eimeria tenella. Both proteins are stage-regulated, and the sporozoite-specific EtAMA1 is effective at inducing partial protection against homologous challenge with E. tenella when used as a recombinant protein vaccine, whereas the merozoite-specific EtAMA2 is not. In order to test the ability of transgenic parasites to confer heterologous protection, E. tenella parasites were complemented with EmAMA1, the sporozoite-specific orthologue of EtAMA1 from E. maxima, coupled with different delivery signals to modify its trafficking and improve antigen exposure to the host immune system. Vaccination of chickens using these transgenic parasites conferred partial protection against E. maxima challenge, with levels of efficacy comparable to those obtained using recombinant protein or DNA vaccines. In the present work we provide evidence for the first known time of the ability of transgenic Eimeria to induce cross protection against different Eimeria spp. Genetically complemented Eimeria provide a powerful tool to streamline the complex multi-valent anticoccidial vaccine formulations that are currently available in the market by generating parasite lines expressing vaccine targets from multiple eimerian species.

Characterization of novel microneme adhesive repeats (MAR) in Eimeria tenella.

BACKGROUND: The phylum Apicomplexa comprises a wide variety of parasites of significant medical and economic relevance. These parasites have extremely different host and tissue tropisms; for example Toxoplasma gondii can invade virtually any nucleated cell and infect almost all warm-blooded vertebrates, whereas Eimeria tenella infects only chickens and is restricted in its growth to epithelial cells of the caecum. Proteins released from the microneme secretory organelles (MICs) are critical for apicomplexan invasion of host cells and allow parasites to bind a diverse range of host cell oligosaccharide epitopes. MICs bear modular arrangements of sequences with adhesive proteins and interestingly the sialic-acid binding MAR (microneme adhesive repeat) domain containing proteins (MCPs) are suggested to make significant contributions to the different host and tissue tropisms of T. gondii and E. tenella. RESULTS: In this study, we evaluated the binding capacity of Type I MAR domains from novel E. tenella MCPs. Variants of the previously described HxT motif were analysed showing that HxT and VxT variants bind, whereas HxS and YxE variants did not. One of these MCP containing a single MAR (EtMCP2) showed an apical localization when expressed as a fusion with the fluorescent reporter mCherry in transgenic populations and a similar pattern of transcripts per zoite during endogenous development in vitro as the well-characterised microneme protein EtMIC2. CONCLUSIONS: Variation in the binding properties of the MAR of different EtMCPs was confirmed and their ability to bind a wider range of sialic acids and terminal linkages should be studied. In addition, transgenesis technology has been used for first time in Eimeria parasites as a rapid tool for the study of endogenous protein localization by fusion with a fluorescent reporter.

Viral proteins expressed in the protozoan parasite Eimeria tenella are detected by the chicken immune system.

BACKGROUND: Eimeria species are parasitic protozoa that cause coccidiosis, an intestinal disease commonly characterised by malabsorption, diarrhoea and haemorrhage that is particularly important in chickens. Vaccination against chicken coccidiosis is effective using wild-type or attenuated live parasite lines. The development of protocols to express foreign proteins in Eimeria species has opened up the possibility of using Eimeria live vaccines to deliver heterologous antigens and function as multivalent vaccine vectors that could protect chickens against a range of pathogens. RESULTS: In this study, genetic complementation was used to express immunoprotective virus antigens in Eimeria tenella. Infectious bursal disease virus (IBDV) causes Gumboro, an immunosuppressive disease that affects productivity and can interfere with the efficacy of poultry vaccination programmes. Infectious laryngotracheitis virus (ILTV) causes a highly transmissible respiratory disease for which strong cellular immunity and antibody responses are required for effective vaccination. Genes encoding the VP2 protein from a very virulent strain of IBDV (vvVP2) and glycoprotein I from ILTV (gI) were cloned downstream of 5'Et-Actin or 5'Et-TIF promoter regions in plasmids that also contained a mCitrine fluorescent reporter cassette under control of the 5'Et-MIC1 promoter. The plasmids were introduced by nucleofection into E. tenella sporozoites, which were then used to infect chickens. Progeny oocysts were sorted by FACS and passaged several times in vivo until the proportion of fluorescent parasites in each transgenic population reached ~20 % and the number of transgene copies per parasite genome decreased to < 10. All populations were found to transcribe and express the transgene and induced the generation of low titre, transgene-specific antibodies when used to immunise chickens. CONCLUSIONS: E. tenella can express antigens of other poultry pathogens that are successfully recognised by the chicken immune system. Nonetheless, further work has to be done in order to improve the levels of expression for its future use as a multivalent vaccine vector.

The tandemly repeated NTPase (NTPDase) from Neospora caninum is a canonical dense granule protein whose RNA expression, protein secretion and phosphorylation coincides with the tachyzoite egress.

BACKGROUND: NTPases (also NTPDases) are enzymes with apyrase activity. They are widely distributed among eukaryotes, and also among members of the family Sarcocystidae. In Toxoplasma gondii, the TgNTPase accumulates in the dense granules, and has been commonly associated with the strain virulence. In the closely related Neospora caninum, the NcNTPase lacks nucleoside diphosphate hydrolase activity and appears to be more abundant in virulent isolates, indicating that it may contribute to the pathogenicity of neosporosis. However, so far no additional information on NcNTPase has been provided. METHODS: Herein, the NcNTPase coding sequences were analysed by different in silico and de novo sequencing approaches. A comparative analysis of NcNTPase and NcGRA7 in terms of protein dynamics, secretion, phosphorylation, and mRNA expression profiles during the tachyzoite lytic cycle was also carried out. Moreover, NcNTPase immunolocalization was analysed by confocal microscopy techniques over a set number of time-points. RESULTS: We describe the presence of three different loci containing three copies of the NcNTPase within the Nc-Liv genome, and report the existence of up to four different NcNTPase alleles in Nc-Liv. We also provide evidence for the occurrence of diverse protein species of the NcNTPase by two-dimensional gel electrophoresis. Both NcNTPase and NcGRA7 were similarly up-regulated and secreted during the egress and/or early invasion phases, and were phosphorylated. However, its secretion was not affected by the addition of calcium modulators such as A23187 and ethanol. NcNTPase and NcGRA7 localized in dense granules and parasitophorous vacuole membrane throughout the lytic cycle, although differed in their inmunolocalization during early invasion and egress. CONCLUSIONS: The present study reveals the complexity of the NcNTPase loci in N. caninum. We hypothesize that the expression of different isoforms of the NcNTPase protein could contribute to parasite virulence. Our findings showed regulation of expression, secretion and phosphorylation of NcNTPase suggesting a potential role for progression through the tachyzoites lytic cycle.