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).

Identification and Characterization of Eimeria tenella Microneme Protein (EtMIC8).

Microneme proteins (MICs) of Eimeria tenella play key roles in motility, migration, attachment, and invasion processes. More than 20 apicomplexan parasite's MICs have been identified, with nine Eimeria MICs being reported. In this study, a novel E. tenella MIC was identified, and its gene structural features, developmental expression levels, localization, role in adhesion and invasion, and immunogenicity were studied. The results showed that the open reading frame was 1,650 bp, encoding 550 amino acids. It contains a signal sequence, a transmembrane region, four low-complexity boxes, and five epidermal growth factor-like domains (EGF). Subcellular localization revealed its distribution on the membrane surface of the parasite. These characteristics are consistent with the common features of MICs and are named EtMIC8. Anti-EtMIC8 antibodies recognized a specific binding of about 100 kDa in E. tenella, which was twice as large as the prokaryotic expression (about 50 kDa), suggesting that MIC8 may exist naturally as a dimer. EtMIC8 was expressed at higher levels in sporozoites (3.08-fold) and merozoites (2.1-fold) than in sporulated oocysts. The attachment assays using a yeast surface display of MIC8 and its different domains showed that the adherence rates of EtMIC8 to host cells were significantly higher than those of the control (3.17-fold), which was the full contribution of EGF, but neither was alone. Anti-EtMIC8 antibodies significantly reduced the invasion rate of sporozoites into host cells compared to those of the control (P < 0.01). Recombinant EtMIC8-EGF peptides could provide moderate protective efficacy (anticoccidial index [ACI]: 169.7), induce humoral responses, and upregulate CD3+CD8+ lymphocyte cells.

Sporulation rate and viability of Eimeria tenella oocysts stored in potassium sorbate solution.

In order to find a new preservation solution for avian coccidial oocysts that can replace potassium dichromate (K2Cr2O7) solution, Eimeria tenella oocysts were preserved in 0.1 to 10% potassium sorbate (C6H7KO2) solution in this study. The results showed that there was no significant difference between the sporulation rate of E. tenella oocysts preserved in 0.1 to 10% C6H7KO2 solution and in 2.5% K2Cr2O7 solution (p > 0.05). The 0.5 to 10% C6H7KO2 solution could also effectively inhibit the growth of bacterial microorganisms. E. tenella oocysts preserved in 1% C6H7KO2 solution at 4 °C for 3, 6, 9, and 12 months, with the oocyst production of E. tenella oocysts being 1.3-, 1.2-, 1.6-, and 1.3-fold higher than that of oocysts stored in 2.5% K2Cr2O7 solution (p < 0.05). In conclusion, C6H7KO2 could replace K2Cr2O7 as the preservation solution of avian coccidial oocysts.

Effect of different floatation solutions on E. tenella oocyst purification and optimization of centrifugation conditions for improved recovery of oocysts and sporocysts.

Saturated salt floatation method is widely used for coccidian oocyst purification. However, the repeated procedures and inefficient oocysts recovery rate are a continuous challenge. This study aimed to investigate the best suitable floatation solution, along with optimal centrifugation speed and time for Eimeria tenella (E. tenella) oocyst and sporocyst purification. Different floatation solutions i-e, saturated salt, Sheather's sugar and sodium hypochlorite (NaClO) at 20-60% concentrations were used to purify oocyst. It was found that about 96.99% oocysts (8609×g for 10 min) were recovered under these conditions without any effect on the viability of sporocysts. The recovery rate of oocysts using 50% NaClO (V/V) was significantly higher than 35% saturated salt flotation solution (P < 0.05). The optimal method for purification of oocysts based our experimentation was centrifugation at 8609×g for 3 min using 50% NaClO floatation solution, and the optimized centrifugation conditions for improved recovery of sporocysts (about 99.3%) were at 2152×g for 5 min. The present study provided a better method for the coccidian oocyst purification, which could be successfully adopted as a better alternative to existing techniques commonly used for investigations/research pertaining to coccidia.

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.

Eimeria tenella Eimeria-specific protein that interacts with apical membrane antigen 1 (EtAMA1) is involved in host cell invasion.

BACKGROUND: Avian coccidiosis is a widespread, economically significant disease of poultry, caused by several Eimeria species. These parasites have complex and diverse life-cycles that require invasion of their host cells. This is mediated by various proteins secreted from apical secretory organelles. Apical membrane antigen 1 (AMA1), which is released from micronemes and is conserved across all apicomplexans, plays a central role in the host cell invasion. In a previous study, some putative EtAMA1-interacting proteins of E. tenella were screened. In this study, we characterized one putative EtAMA1-interacting protein, E. tenella Eimeria -specific protein (EtEsp). METHODS: Bimolecular fluorescence complementation (BiFC) and glutathione S-transferase (GST) fusion protein pull-down (GST pull-down) were used to confirm the interaction between EtAMA1 and EtEsp in vivo and in vitro. The expression of EtEsp was analyzed in different developmental stages of E. tenella with quantitative PCR and western blotting. The secretion of EtEsp protein was tested with staurosporine when sporozoites were incubated in complete medium at 41 °C. The localization of EtEsp was analyzed with an immunofluorescence assay (IFA). An in vitro invasion inhibition assay was conducted to assess the ability of antibodies against EtEsp to inhibit cell invasion by E. tenella sporozoites. RESULTS: The interaction between EtAMA1 and EtEsp was confirmed with BiFC and by GST pull-down. Our results show that EtEsp is differentially expressed during distinct phases of the parasite life-cycle. IFA showed that the EtEsp protein is mainly distributed on the parasite surface, and that the expression of this protein increases during the development of the parasite in the host cells. Using staurosporine, we showed that EtEsp is a secreted protein, but not from micronemes. In inhibition tests, a polyclonal anti-rEtEsp antibody attenuated the capacity of E. tenella to invade host cells. CONCLUSION: In this study, we show that EtEsp interacts with EtAMA1 and that the protein is secreted protein, but not from micronemes. The protein participates in sporozoite invasion of host cells and is maybe involved in the growth of the parasite. These data have implications for the use of EtAMA1 or EtAMA1-interacting proteins as targets in intervention strategies against avian coccidiosis.

The Effect of Increased Temperatures on Viability, Morphology, Infectivity, and Development of Eimeria tenella.

Commonly found in backyard and commercial poultry production, coccidiosis, caused by Eimeria species, presents a self-limiting intestinal infection based on the number of ingested oocysts. Heat stress (HS) is one of the major environmental stressors in poultry, predisposing broiler chickens to immunosuppression and rendering them susceptible to diseases. There are suggestions that HS reduces Eimeria oocyst shedding in chickens; however, the relationship between HS and coccidiosis is not well elucidated. The objective of this study was to investigate the effect of temperature on viability, morphology, infectivity, and development of Eimeria tenella in vitro, and merozoite production and oocyst shedding in vivo. In vitro exposure of sporozoites to 55 C for at least 60 min reduced sporozoites viability as shown by morphological changes and rendering them unable to invade Mardin-Darbi bovine kidney (MDBK) cells. Intracellular development of merozoites was significantly reduced by an increase in 2 C in the optimal temperature of incubation in vitro. Most importantly, the induction of HS in the live chickens caused significantly lower lesion scores, reduced merozoite production, and oocyst shedding, resulting in a much less severe disease outcome.

Proteomic analysis of the second-generation merozoites of Eimeria tenella under nitromezuril and ethanamizuril stress.

BACKGROUND: Eimeria tenella is a highly pathogenic coccidian that causes avian coccidiosis. Both nitromezuril (NZL) and ethanamizuril (EZL) are novel triazine compounds with high anticoccidial activity, but the mechanisms of their action are still unclear. This study explored the response of E. tenella to NZL and EZL by the study of changes in protein composition of the second-generation merozoites. METHODS: Label-free quantification (LFQ) proteomics of the second-generation merozoites of E. tenella following NZL and EZL treatment were studied by LC-MS/MS to explore the mechanisms of action. The identified proteins were annotated and analyzed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and protein-protein interaction (PPI) networks analysis. RESULTS: A total of 1430 proteins were identified by LC-MS/MS, of which 375 were considered as differential proteins in response to drug treatment (DPs). There were 26 only found in the NZL treatment group (N-group), 63 exclusive to the EZL treatment group (E-group), and 80 proteins were present in both drug groups. In addition, among the DPs, the abundant proteins with significantly altered expression in response to drug treatment (SDPs) were found compared with the C-group, of which 49 were upregulated and 51 were downregulated in the N-group, and 66 upregulated and 79 downregulated in the E-group. Many upregulated proteins after drug treatment were involved in transcription and protein metabolism, and surface antigen proteins (SAGs) were among the largest proportion of the downregulated SDPs. Results showed the top two enriched GO terms and the top one enriched pathway treated with EZL and NZL were related, which indicated that these two compounds had similar modes of action. CONCLUSIONS: LFQ proteomic analysis is a feasible method for screening drug-related proteins. Drug treatment affected transcription and protein metabolism, and SAGs were also affected significantly. This study provided new insights into the effects of triazine anticoccidials against E. tenella.

Excystation of Eimeria acervulina, E. maxima, and E. tenella differs in response to trypsin and chymotrypsin and the presence of reducing agents DTT and TCEP.

Release of sporozoites from Eimeria oocysts/sporocysts is an essential step in the intracellular development of the parasite in its host. Little is known about this process except that elevated temperature (∼ 40 °C) plus trypsin and bile salts are required for sporozoite to escape from sporocysts. In this study, it was found that adding a reducing agent, either dithiothreitol (DTT) or Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), increased the lifespan of sporozoites released from Eimeria maxima. While the addition of DTT or TCEP affected the apparent molecular weight of trypsin, it did not interfere with excystation of E. maxima, but rather had a positive effect on the number of viable sporozoites present after release. This effect was time-dependent in that the number of intact sporozoites at 15 and 30 min after excystation was similar between untreated and DTT- or TCEP-treated sporocysts. However, by 45-60 min, virtually no sporozoites were observed in excystation fluid not containing DTT or TCEP. Of interest is that this effect appeared to be Eimeria species-dependent. Eimeria acervulina and E. tenella sporozoites remained viable for at least 60 min after excystation in the absence of DTT or TCEP. The effect of DTT and TCEP on chymotrypsin was also studied with all 3 Eimeria species because there is some evidence that chymotrypsin is an effective excystation enzyme. Indeed, E. maxima sporozoites excysting from sporocysts with chymotrypsin in the presence of DTT or TCEP remained viable for at least 60 min after release, unlike excystation done in the absence of these reducing agents. Chymotrypsin was capable of excysting E. acervulina in the presence or absence of DTT or TCEP. Of interest, is that chymotrypsin was ineffective in the excystation of E. tenella. These findings suggest that trypsin and chymotrypsin have differential effects on sporozoite excystation and that reducing agents may alter sites on the enzyme that affect sporozoite viability, but not release from sporocysts.

Molecular characterization of surface antigen 10 of Eimeria tenella.

Chicken coccidiosis is caused by the apicomplexan parasite Eimeria spp. At present, drug resistance of Eimeria is common because of the indiscriminate use of anticoccidial drugs. The gene encoding surface antigen 10 of Eimeria tenella (EtSAG10) is differentially expressed between drug-resistant and drug-sensitive strains. RNA-seq analysis indicated that this gene was downregulated in strains resistant to maduramicin and diclazuril compared to susceptible strains. EtSAG10 DNA sequence alignment revealed that they contained one and ten mutations in MRR and DZR, compared with DS, respectively. A full-length EtSAG10 cDNA was successfully cloned and expressed, and the polyclonal antibody was prepared. The transcription and translation levels of EtSAG10 were analyzed by quantitative real-time PCR (qPCR) and Western blotting. The localization of EtSAG10 in Spz, Mrz, and parasites in the first asexual stage was determined by indirect immunofluorescence. The potential association of EtSAG10 with sporozoite invasion of host cells was assessed by invasion inhibition assays. The results showed that EtSAG10 had a predicted transmembrane domain at the C-terminal end and a predicted signal peptide at the N-terminal end. EtSAG10 was downregulated in drug-resistant strains, which is consistent with the RNA-seq results. The EtSAG10 protein was localized to the parasite surface and parasitophorous vacuole membrane. This protein was shown to play a role in the infection of chicken intestine by sporozoites.

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.

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.

Eimeria tenella protein trafficking: differential regulation of secretion versus surface tethering during the life cycle.

Eimeria spp. are intracellular parasites that have a major impact on poultry. Effective live vaccines are available and the development of reverse genetic technologies has raised the prospect of using Eimeria spp. as recombinant vectors to express additional immunoprotective antigens. To study the ability of Eimeria to secrete foreign antigens or display them on the surface of the sporozoite, transiently transfected populations of E. tenella expressing the fluorescent protein mCherry, linked to endogenous signal peptide (SP) and glycophosphatidylinositol-anchor (GPI) sequences, were examined. The SP from microneme protein EtMIC2 (SP2) allowed efficient trafficking of mCherry to cytoplasmic vesicles and following the C-terminal addition of a GPI-anchor (from surface antigen EtSAG1) mCherry was expressed on the sporozoite surface. In stable transgenic populations, mCherry fused to SP2 was secreted into the sporocyst cavity of the oocysts and after excystation, secretion was detected in culture supernatants but not into the parasitophorous vacuole after invasion. When the GPI was incorporated, mCherry was observed on the sporozites surface and in the supernatant of invading sporozoites. The proven secretion and surface exposure of mCherry suggests that antigen fusions with SP2 and GPI of EtSAG1 may be promising candidates to examine induction of protective immunity against heterologous pathogens.

Dynamic changes in the main regulatory genes of mitochondrial permeability transition pore in Eimeria tenella host cells.

The purpose of the present study was to investigate the dynamic changes in the main regulatory genes of the mitochondrial permeability transition pore in E. tenella host cells. Primary chick embryo cecum epithelial cell culture techniques, spectrophotometer technology, Hoechst-Annexin V-PI apoptosis staining and ELISA were used to detect the apoptosis rate and dynamic changes of Bcl-2, Bcl-xl, Bax, Bak, Bid, Bad, HK-II, and ATP content in E. tenella host cells at 4, 24, 48, 72, 96, and 120 h. The rates of early apoptosis, late apoptosis, and necrosis of group T0 were significantly lower (P < 0.05) or highly significantly lower (P < 0.01) than those of group C at 4 h, but higher (P < 0.05 or P < 0.01) at varying degrees than those of the same group at 24-120 h. Compared to group C, the amount of Bcl-2, ATP, Bax and Bad in group T0 were visibly lower (P < 0.05 or P < 0.01) at 4 h, whereas Bcl-xl/Bax was highly significantly higher (P < 0.01) at 4 h. In addition, group T0 had less ATP at 24-120 h than group C, whereas the amount of Bcl-2, Bcl-xl, Bax, Bak, Bid, Bad and HK-II in group T0 inversely increased in varying degrees at 24-120 h compared with group C. Moreover, Bcl-2/Bax was lower (P < 0.01) at 24, 48, and 96 h, and Bcl-xl/Bax was lower (P < 0.05) at 48 h in group T0 than in group C, respectively. Taken together, these observations indicate that in the early developmental stages of E. tenella, the host-cell apoptosis rate decreased; although the amount of anti- and pro-apoptotic genes in host cells decreased, the ratios of anti-apoptotic to pro-apoptotic bcl-2 gene-family members increased. In the middle and later developmental stages of E. tenella, the host-cell apoptosis rate increased; the amount of anti- and pro-apoptotic genes increased, while the ratios of anti-apoptotic to pro-apoptotic bcl-2 gene-family members decreased. In addition, ATP decreased at all developmental stages of E. tenella.

Expression of perforin, granzyme A and Fas ligand mRNA in caecal tissues upon Eimeria tenella infection of naïve and immune chickens.

Cytotoxic cells of the immune system may kill infected or transformed host cells via the perforin/granzyme or the Fas ligand (FasL) pathways. The purpose of this study was to determine mRNA expression of perforin, granzyme A and FasL in Eimeria tenella-infected tissues at primary infection and infection of immune chickens as an indirect measure of cytotoxic cell activity. Chickens were rendered immune by repeated E. tenella infections, which were manifested as an absence of clinical signs or pathological lesions and significantly reduced oocyst production upon challenge infection. During primary E. tenella infection, perforin, granzyme A and FasL mRNA expression in caecal tissue was significantly increased at 10 days after infection, compared to uninfected birds. In contrast, at infection of immune birds, perforin and granzyme A mRNA expression in caecal tissue was significantly increased during the early stages of E. tenella challenge infection, days 1-4, which coincided with a substantial reduction of parasite replication in these birds. These results indicate the activation of cytotoxic pathways in immune birds and support a role for cytotoxic T cells in the protection against Eimeria infections.

Transcriptional profiles of virulent and precocious strains of Eimeria tenella at sporozoite stage; novel biological insight into attenuated asexual development.

Chicken coccidiosis is caused by Eimeria spp., particularly Eimeria tenella, and is characterized by watery or hemorrhagic diarrhea, resulting in death in severe cases. Precociously attenuated live vaccines are widely used to control the disease, and these are produced by serially passaging virulent strains through chickens, and the collection of oocysts from feces at progressively earlier time points during oocyst shedding. Sporozoites of the precocious strain rapidly enter the intestinal mucosa, and their subsequent asexual development reduces their growth. However, there have been few detailed genetic or transcriptional analyses of the strains. Here, we used RNA sequencing to gain novel biological insight into the pathogenicity and precocity of E. tenella. We compared the differential transcription in the sporozoites (the initial stage of endogenous development) of virulent and precocious strains by mapping the sequence reads onto the draft genome of E. tenella. About 90% of the reads from both strains were mapped to the genome, and 16,630 estimated transcript regions were identified. Using Gene Ontology slim and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses and the annotation of the estimated transcripts with Blastx, we found that the expression of some genes involved in carbohydrate metabolism were expressed two-fold more strongly in the virulent strain than in the precocious strain. Characteristically, genes related to proteins secreted from the apical complex, proteases, cell attachment proteins, mitochondrial proteins, and transporters were most strongly upregulated in the virulent strain. Interestingly, the expression of genes associated with cell survival, development, or proliferation was strongly upregulated in the precocious strain. These findings suggest that virulent strains survive long before invasion and invade actively/successfully into host cells, whereas proliferative processes appear to affect precocity.

Ultrastructural effects of acetamizuril on endogenous phases of Eimeria tenella.

To explore the primary stage or site of action of acetamizuril (AZL), a novel triazine anticoccidial compound, the ultrastructural development of Eimeria tenella at different endogenous stages was studied in experimentally infected chickens treated with a single oral dose of 15 mg/kg AZL. As a result of drug action, the differentiations of second-generation schizonts and microgamonts were largely inhibited and merozoites became irregular in shape. Meanwhile, the outer membrane blistering and perinuclear space enlargement were obvious in the second-generation schizonts and microgamonts, which were never observed in the classic triazine anticoccidiosis drug diclazuril-treated E. tenella. The chromatin aggregation, anachromasis, and marginalization were visible in merozoites and microgamonts. Furthermore, the abnormal evagination of microgametes finally resulted in the degeneration of microgamonts and the failure of subsequent fertilization. The most marked micromorphological alteration occurring in the macrogamonts was the WFB2. Even if the fertilization occurred, the formation of oocyst wall became malformed and the zygote proceeded to the obvious degeneration. In addition, mitochondria swelling and cytoplasm vacuolization were discerned in respective intracellular stages, while endoplasmic reticulum and Golgi body swelling was less seen. These alterations may be the causes leading to the final death of E. tenella and also provide some information for further exploring the mechanism of action of AZL at the molecular level.

In vitro efficacy of allicin on chicken Eimeria tenella sporozoites.

Chicken coccidiosis is a major parasitic disease caused by Eimeria spp. It is controlled and treated using chemical anticoccidial agents. Development of partial or complete resistance toward these anticoccidials is considered a major problem in poultry industry. Allicin is an organosulfur compound produced as a result of the reaction between alliin and alliinase after hacking of garlic. In this study, tenfold dilution from 180 mg/ml to 1.8 ng/ml of allicin in distilled water was tested against E. tenella in vitro. The percent of inhibition in allicin was from 99.9 to 71.53% using 180 mg/ml and 180 ng/ml, respectively. The percent of inhibition was 56.24% using 1.8 ng/ml. We used allicin as a treatment from plants against chicken coccidiosis; however, in vivo study should be performed to confirm these results.

Cloning and characterization of three Eimeria tenella lipid phosphate phosphatases.

Although lipid phosphate phosphatases (LPPs) play an important role in cellular signaling in addition to lipid biosynthesis, little is thus far known about parasite LPPs. In this study, we characterized three Eimeria tenella cDNA clones encoding LPP named EtLPP1, EtLPP2 and EtLPP3. Key structural features previously described in LPPs, including the three conserved domains proposed as catalytic sites, a single conserved N-glycosylation site, and putative transmembrane domains were discovered in the three resulting EtLPP amino acid sequences. Expression of His6-tagged EtLPP1, -2, and -3 in HEK293 cells produced immunoreactive proteins with variable molecular sizes, suggesting the presence of multiple forms of each of the three EtLPPs. The two faster-migrating protein bands below each of the three EtLPP proteins were found to be very similar to the porcine 35-kDa LPP enzyme in their molecular size and the extent of their N-glycosylation, suggesting that the three EtLPPs are partially N-glycosylated. Kinetic analyses of the activity of the three enzymes against PA, LPA, C1P and S1P showed that Km values for each of the substrates were (in µM) 284, 46, 28, and 22 for EtLPP1; 369, 179, 237, and 52 for EtLPP2; and 355, 83, and 260 for EtLPP3. However, EtLPP3 showed negligible activity on S1P. These results confirmed that the three EtLPPs have broad substrate specificity. The results also indicated that despite structural similarities, the three EtLPPs may play distinct functions through their different models of substrate preference. Furthermore, particularly high expression levels of the three EtLPP genes were detected in the sporozoite stage of the E. tenella life cycle (p<0.001), suggesting that their encoded proteins might play an important biological function in the sporozoite stage.

RNA Seq analysis of the Eimeria tenella gametocyte transcriptome reveals clues about the molecular basis for sexual reproduction and oocyst biogenesis.

BACKGROUND: The protozoan Eimeria tenella is a common parasite of chickens, causing avian coccidiosis, a disease of on-going concern to agricultural industries. The high prevalence of E. tenella can be attributed to the resilient oocyst stage, which is transmitted between hosts in the environment. As in related Coccidia, development of the eimerian oocyst appears to be dependent on completion of the parasite's sexual cycle. RNA Seq transcriptome profiling offers insights into the mechanisms governing the biology of E. tenella sexual stages (gametocytes) and the potential to identify targets for blocking parasite transmission. RESULTS: Comparisons between the sequenced transcriptomes of E. tenella gametocytes and two asexual developmental stages, merozoites and sporozoites, revealed upregulated gametocyte transcription of 863 genes. Many of these genes code for proteins involved in coccidian sexual biology, such as oocyst wall biosynthesis and fertilisation, and some of these were characterised in more depth. Thus, macrogametocyte-specific expression and localisation was confirmed for two proteins destined for incorporation into the oocyst wall, as well as for a subtilisin protease and an oxidoreductase. Homologues of an oocyst wall protein and oxidoreductase were found in the related coccidian, Toxoplasma gondii, and shown to be macrogametocyte-specific. In addition, a microgametocyte gamete fusion protein, EtHAP2, was discovered. CONCLUSIONS: The need for novel vaccine candidates capable of controlling coccidiosis is rising and this panel of gametocyte targets represents an invaluable resource for development of future strategies to interrupt parasite transmission, not just in Eimeria but in other Coccidia, including Toxoplasma, where transmission blocking is a relatively unexplored strategy.