Glyceraldehyde-3-phosphate dehydrogenase from Eimeria acervulina modulates the functions of chicken dendritic cells to boost Th1 type immune response and stimulates autologous CD4+ T cells differentiation in-vitro.

Dendritic cells (DCs) play a pivotal role to amplify antigen-specific immune responses. Antigens that sensitize T cells via antigen-presentation by DCs could enhance the capacity of host immunity to fight infections. In this study, we tested the immunogenic profiles of chicken DCs towards Glyceraldehyde-3-phosphate dehydrogenase from Eimeria acervulina (EaGAPDH). Immunoblot analysis showed that recombinant EaGAPDH (rEaGAPDH) protein was successfully recognized by rat sera generated against rEaGAPDH. Interaction and internalisation of rEaGAPDH by chicken splenic-derived DCs (chSPDCs) was confirmed by immunofluorescence analysis. Flow cytometry revealed that chSPDCs upregulated MHCII, CD1.1, CD11c, CD80, and CD86 cell-surface markers. Moreover, mRNA expressions of DC maturation biomarkers (CCL5, CCR7, and CD83) and TLR signalling genes (TLR15 and MyD88) were also upregulated whereas those of Wnt signalling were non-significant compared to negative controls. rEaGAPDH treatment induced IL-12 and IFN-γ secretion in chSPDCs but had no effect on IL-10 and TGF-ß. Likewise, DC-T cell co-culture promoted IFN-γ secretion and the level of IL-4 was unaffected. Proliferation of T cells and their differentiation into CD3+/CD4+ T cells were triggered in chSPDCs-T cells co-culture system. Taken together, rEaGAPDH could promote Th1 polarization by activating both host DCs and T cells and sheds new light on the role of this important molecule which might contribute to the development of new DCs-based immunotherapeutic strategies against coccidiosis.

Recombinant ubiquitin-conjugating enzyme of Eimeria maxima induces immunogenic maturation in chicken splenic-derived dendritic cells and drives Th1 polarization in-vitro.

Dendritic cells (DCs) are key linkages between innate immunity and acquired immunity. The antigens that promote the functions of DCs might be the effective candidates of novel vaccine. In this research, the ability of ubiquitin-conjugating enzyme (UCE), a recognized common antigens among chicken Eimeria species, to stimulate DCs of chickens were evaluated. We cloned UCE gene from Eimeria maxima (EmUCE), and its protein expression was confirmed by SDS-PAGE and western-blot. Immunofluorescence assay confirmed the binding of rEmUCE on the surface of chicken splenic-derived DCs (ChSP-DCs). Flow cytometric analysis showed that rEmUCE-treated ChSP-DCs increased MHCII, CD1.1, CD11c, CD80, and CD86 phenotypes. qRT-PCR indicated that transcript levels of maturation markers CCL5, CCR7, and CD83 in ChSP-DCs were upregulated in response to rEmUCE. Following rEmUCE treatment, chSP-DCs activated TLR signaling and inhibited Wnt signaling. Moreover, rEmUCE promoted DC-mediated T-cell proliferation in DC/T-cell co-incubation. Interestingly, CD3+/CD4+ T-cells were significantly enhanced when rEmUCE-treated chSP-DCs were co-incubated with T-cells. Cytokine secretion pattern of rEmUCE-stimulated ChSP-DCs revealed that the production of IL-12 and IFN-γ was increased whereas IL-10 and TGF-ß were unchanged. Likewise, the co-incubation of ChSP-DCs with T-cells indicated increased production of IFN-γ but not IL-4. Collectively, rEmUCE could polarize DCs to immunogenic phenotype and shift the immune cells towards Th1 response. Our observations provide valuable insight for future research aimed at vaccine development against avian coccidiosis.

An Eimeria acervulina OTU protease exhibits linkage-specific deubiquitinase activity.

Ubiquitination is an important post-translational modification process that regulates many cellular processes. Proteins can be modified at single or multiple lysine residues by a single ubiquitin protein or by ubiquitin oligomers. It is important to note that the type of ubiquitin chains determines the functional outcome of the modification. Ubiquitin or ubiquitin chains can be removed by deubiquitinases (DUBs). In our previous study, the Eimeria tenella ovarian tumour (Et-OTU) DUB was shown to regulate the telomerase activity of E. tenella and affect E. tenella proliferation. The amino acid sequences of Et-OTU (GenBank: XP_013229759.1) and Eimeria acervulina (E. acervulina) ovarian tumour (Ea-OTUD3) DUB (XP_013250378.1) are 74% identical. Although Et-OTU may regulate E. tenella telomerase activity, whether Ea-OTUD3 affects E. acervulina growth and reproduction remains unclear. We show here that Ea-OTUD3 belongs to the OTU domain class of cysteine protease deubiquitinating enzymes. Ea-OTUD3 is highly linkage-specific, cleaving K48 (Lys48)-, K63-, and K6-linked diubiquitin but not K29-, K33-, and K11-linked diubiquitin. The precise linkage preference of Ea-OTUD3 among these three nonlinear diubiquitin chains is K6 > K48 > K63. Recombinant Ea-OTUD3, but not its catalytic-site mutant Ea-OTUD3 (C247A), exhibits activity against diubiquitin. Ea-OTUD3 removes ubiquitin from the K48-, but to a lesser extent from the K63-linked ubiquitinated E. acervulina proteins of the modified target protein, thereby exhibiting the characteristics of deubiquitinase. This study reveals that the Ea-OTUD3 is a novel functional deubiquitinating enzyme. Furthermore, the Ea-OTUD3 protein may regulate the stability of some K48-linked ubiquitinated E. acervulina proteins.

Molecular detection of field isolates of Turkey Eimeria by polymerase chain reaction amplification of the cytochrome c oxidase I gene.

Oocysts of Eimeria spp. were isolated from litter samples obtained from 30 commercial turkey farms. Genomic DNA was extracted from clean oocysts, and polymerase chain amplification of the species-specific cytochrome c oxidase subunit I (COI) gene was performed for five species of turkey Eimeria. The species tested were Eimeria adenoeides, Eimeria meleagrimitis, Eimeria meleagridis, Eimeria dispersa, and Eimeria gallopavonis. All DNA samples were positive for E. meleagrimitis, nine were positive for E. adenoeides, two were positive for E. dispersa, and none for E. meleagridis and E. gallopavonis. E. meleagrimitis occurred as a single species in 21 (70 %) of the farms while 9 (30 %) farms had a mixed species with E. meleagrimitis and E. adenoeides and 2 (7 %) were triple positive with E. meleagrimitis, E. adenoeides, and E. dispersa. This is the first account of the field prevalence of turkey Eimeria species using molecular methods.

Some heterocyclic thione derivatives exhibit anticoccidial activity by inhibiting glycosidases.

Coccidiosis is one of the most common parasitic diseases affecting many species of domestic animals. This disease has a major economic significance and the search for new compounds having anticoccidial activity is of great importance. In this article, different levels of protection from coccidian infection by Eimeria stiedae were developed in rabbits by treatment with compounds incorporating the skeleton of thiourea. These compounds include 4,5-diphenylimidazole-2-thione (1), 4,5-Diphenyl-1,2,4-triazole-3-thiol (2) and 5-(2-Hydroxyphenyl)-4-phenyl-1,2,4-triazole-3-thiol (3) compared to the anticoccidial drug toltrazuril as a reference compound. Compounds 1-3 inhibit coccidiosis-induced activity of α-glucosidase. The protection from coccidial infection by compound 1 was higher than that shown for compounds 2 and 3. These data suggest that diazole and triazole thione derivatives have a mimetic effect for anticoccidial drugs through their inhibition of glycosidases.

Peroxidase catalysed cross-linking of an intrinsically unstructured protein via dityrosine bonds in the oocyst wall of the apicomplexan parasite, Eimeria maxima.

Apicomplexan parasites such as Eimeria maxima possess a resilient oocyst wall that protects them upon excretion in host faeces and in the outside world, allowing them to survive between hosts. The wall is formed from the contents of specialised organelles - wall-forming bodies - found in macrogametes of the parasites. The presence of dityrosine in the oocyst wall suggests that peroxidase-catalysed dityrosine cross-linking of tyrosine-rich proteins from wall-forming bodies forms a matrix that is a crucial component of oocyst walls. Bioinformatic analyses showed that one of these tyrosine-rich proteins, EmGAM56, is an intrinsically unstructured protein, dominated by random coil (52-70%), with some α-helix (28-43%) but a relatively low percentage of ß-sheet (1-11%); this was confirmed by nuclear magnetic resonance and circular dichroism. Furthermore, the structural integrity of EmGAM56 under extreme temperatures and pH indicated its disordered nature. The intrinsic lack of structure in EmGAM56 could facilitate its incorporation into the oocyst wall in two ways: first, intrinsically unstructured proteins are highly susceptible to proteolysis, explaining the several differently-sized oocyst wall proteins derived from EmGAM56; and, second, its flexibility could facilitate cross-linking between these tyrosine-rich derivatives. An in vitro cross-linking assay was developed using a recombinant 42kDa truncation of EmGAM56. Peroxides, in combination with plant or fungal peroxidases, catalysed the rapid formation of dityrosine cross-linked polymers of the truncated EmGAM56, as determined by western blotting and HPLC, confirming this protein's propensity to form dityrosine bonds.

Eimeria maxima phosphatidylinositol 4-phosphate 5-kinase: locus sequencing, characterization, and cross-phylum comparison.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) may play an important role in host-cell invasion by the Eimeria species, protozoan parasites which can cause severe intestinal disease in livestock. Here, we report the structural organization of the PIP5K gene in Eimeria maxima (Weybridge strain). Two E. maxima BAC clones carrying the E. maxima PIP5K (EmPIP5K) coding sequences were selected for shotgun sequencing, yielding a 9.1-kb genomic segment. The EmPIP5K coding region was initially identified using in silico gene-prediction approaches and subsequently confirmed by mapping rapid amplification of cDNA ends and RT-PCR-generated cDNA sequence to its genomic segment. The putative EmPIP5K gene was located at position 710-8036 nt on the complimentary strand and comprised of 23 exons. Alignment of the 1147 amino acid sequence with previously annotated PIP5K proteins from other Apicomplexa species detected three conserved motifs encompassing the kinase core domain, which has been shown by previous protein deletion studies to be necessary for PIP5K protein function. Phylogenetic analysis provided further evidence that the putative EmPIP5K protein is orthologous to that of other Apicomplexa. Subsequent comparative gene structure characterization revealed events of intron loss/gain throughout the evolution of the apicomplexan PIP5K gene. Further scrutiny of the genomic structure revealed a possible trend towards "intron gain" between two of the motif regions. Our findings offer preliminary insights into the structural variations that have occurred during the evolution of the PIP5K locus and may aid in understanding the functional role of this gene in the cellular biology of apicomplexan parasites.

Changes of cytokines and IgG antibody in chickens vaccinated with DNA vaccines encoding Eimeria acervulina lactate dehydrogenase.

The aim of this study was to investigate the changes of cytokines and specific serum IgG in chickens following vaccination with DNA vaccines encoding either Eimeria acervulina (E. acervulina) lactate dehydrogenase (LDH) antigen or LDH and chicken IL-2 or IFN-γ. Two-week-old chickens were randomly divided into five groups. Experimental group of chickens were immunized with DNA vaccines while control group of chickens were injected with pVAX1 plasmid alone or sterile water. All immunizations were boosted 2 weeks later. The LDH-specific IgG antibody response was measured at weeks 1-6 post-second immunization. The result showed that the antibody titers in chickens vaccinated with DNA vaccines were significantly different from those of the control groups 1 week after the second immunization (P<0.05) and reached the maximum values 3 weeks post-second immunization. The systemic and local cytokine mRNA expression was determined by quantitative RT-PCR 7 days post-second immunization. The specific IgG antibody levels against LDH of all chickens vaccinated with vaccines were increased compared to those of sterile water (H(2)O) and plasmid (pVAX1) control chickens 1-6 weeks post-second immunization (P<0.05). The mRNA levels of IFN-γ, IL-2, TNFSF15, IL-17D as well as TGF-ß4 in both spleen and cecal tonsil were also increased in experimental chickens. In contrast, the only significant change of IL-4 mRNA level was observed in spleen of chickens immunized with pVAX-LDH-IL-2 compared with pVAX-LDH and control groups (P<0.05). These results suggested that DNA vaccines could increase the IgG antibody level and induce the expressions of cytokines.

Efficacy of DNA vaccines carrying Eimeria acervulina lactate dehydrogenase antigen gene against coccidiosis.

The efficacies of DNA vaccines encoding either Eimeria acervulina lactate dehydrogenase (LDH) antigen or a combination of LDH antigen and chicken IL-2 or IFN-gamma were evaluated against chicken coccidiosis. Three vaccine plasmids pVAX-LDH, pVAX-LDH-IFN-gamma and pVAX-LDH-IL-2 were constructed using the eukaryotic expression vector pVAX1. Expressions of proteins encoded by plasmids DNA in vivo were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay. Average body weight gain, oocyst output, survival rate and lesion scores were measured to evaluate the protective effects of vaccination on challenge infection. The results showed that DNA vaccines could obviously alleviate body weight loss, duodenal lesions, oocyst output and enhance oocyst decrease ratio. Anti-coccidial indexes (ACIs) of pVAX-LDH-IFN-gamma and pVAX-LDH-IL-2 groups were higher than that of other groups. Flow cytometric analysis of T lymphocytes in spleen and cecal tonsil demonstrated that DNA vaccines had significantly increased percentages of CD3(+) T cells compared with pVAX1 alone or TE buffer. The results provided the first proof that DNA vaccine carrying E. acervulina LDH antigen gene induced protective immunity against homologous infection and its effect could be enhanced by co-expression of chicken IL-2 or IFN-gamma.

N-alkyl-4-piperidinyl-2,3-diarylpyrrole derivatives with heterocyclic substitutions as potent and broad spectrum anticoccidial agents.

Diaryl-(4-piperidinyl)-pyrrole derivatives bearing cyclic amine substituents have been synthesized and evaluated as anticoccidial agents. Improvements in potency of Et-PKG inhibition, such as azetidine derivative 3a, and broad spectrum anticoccidial activities in feed, such as morpholine derivative 8c, have been achieved.

RNA-dependent RNA polymerase activity associated with virus-like dsRNA in Eimeria maxima and E. necatrix of the domestic fowl.

RNA-dependent RNA polymerase (RDRP) activity was identified in lysates of Eimeria maxima sporozoites and E. necatrix sporozoites and merozoites. Pretreatment of cell lysates with DNase I, RNase A, proteinase K and actinomycin D prior to RDRP assay was employed to characterize RDRP activity. DNase I and actinomycin D had little effect, while proteinase K abolished RDRP activity in both species. RNase A at a concentration of 1 mg/ml also reduced the polymerase activity in E. maxima and E. necatrix sporozoite lysates to 2% and 0%, respectively. Gel electrophoresis of RDRP products revealed that while most migrated at sizes less than 3 kb, a proportion of labelled products of E. necatrix and E. maxima also migrated to the sizes of their respective putative viral genomes. The RDRP products of E. necatrix were shown to be single-stranded by digestion with RNase in both low- and high-salt solutions and by methylmercuric hydroxide treatment. Moreover, the RDRP products of E. necatrix only hybridized to the 5.6-kb dsRNA of E. necatrix but not to the 4.5-kb dsRNAs of E. necatrix or E. maxima.

Purification of a leucine aminopeptidase from Eimeria falciformis.

A leucine aminopeptidase was purified from the oocysts of Eimeria falciformis using affinity chromatography and gel filtration techniques. It had a molecular weight of 45-50 kDa. Its maximal activity against leucyl-p-nitro anilide was at pH 8.6. It is a metallo-enzyme highly inhibited by bestatin.

Isoenzymes of Eimeria from the domestic fowl: electrophoretic variants among species, strains and clones.

Electrophoretic variation of enzymes in five Eimeria spp. of the domestic fowl, including nine strains, ten single-sporocyst clones and two single-sporozoite clones of E. acervulina, three strains each of E. maxima and E. tenella, two strains of E. praecox and one strain of E. necatrix, were assayed using cellulose acetate electrophoresis. Ten enzymes [aldehyde oxidase (AO), alkaline phosphatase (ALP), amylase (AMY), fumarate hydratase (FUM), glucose-6-phosphate dehydrogenase (G6PDH), glucose phosphate isomerase (GPI), glutamate-oxaloacetate transferase (GOT), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH) and phosphoglucomutase (PGM)] were analyzed for their ability to distinguish between these species and strains. Enzymatic activity of G6PDH, GPI, IDH, MDH and PGM was detected in all the Eimeria spp. examined. Strains within each species were characterized by the same electrophoretic variant of G6PDH. Electrophoretic variants of GPI and PGM were the most valuable in the identification of inter- and intra-specific variation, particularly in the field strains of E. acervulina and E. tenella. These two enzymes were used to examine single-sporocyst and single-sporozoite clones derived from two strains of E. acervulina. The enzymes in E. maxima appeared to be conserved, showing no variation among strains with the five enzymes detected. Relative mobilities, calculated as described in this paper, were found to be consistent between different electrophoresis runs and may serve as a reference when this medium is used.

Sequence, expression and localization of calmodulin-domain protein kinases in Eimeria tenella and Eimeria maxima.

We have isolated and sequenced cDNA clones from Eimeria tenella and Eimeria maxima which encode proteins that share homology with a recently described family of calmodulin-domain protein kinases. The primary sequence data show that each of the protein kinases can be divided into 2 main functional domains-an amino-terminal catalytic domain typical of serine/threonine protein kinases and a carboxy-terminal domain homologous to calmodulin, which is capable of binding calcium ions at 4 'EF-hand' motifs. Expression of the E. tenella calmodulin-domain protein kinase (EtCDPK) increased towards the end of oocyst sporulation, as judged by Northern and Western blotting, and indirect immunofluorescent antibody labelling showed that within a few minutes of adding sporozoites to target host cells in in vitro culture EtCDPK was found to be specifically associated with a filament-like structure that converges at the apical end of the parasite. Once the parasite entered the host cell EtCDPK appeared to be left on the host cell membrane at the point of entry, indicating a brief yet specific role for this molecule in the invasion of host cells by E. tenella.

Cloning and characterization of an Eimeria acervulina sporozoite gene homologous to aspartyl proteinases.

A lambda ZapII cDNA library was constructed using mRNA from Eimeria acervulina sporulated oocysts and screened with monoclonal antibodies raised against Eimeria tenella sporulated oocytes. Monoclonal antibody N3C8B12 identified a clone (6S2) potentially encoding an aspartyl proteinase since significant homology with cathepsin D, pepsin and renin proteinases was revealed by sequence comparisons. The 1500-bp cDNA fragment containing the coccidial gene was subcloned into pGEX-FA expression vector, leading to the production of an 80-kDa fusion protein (FA6S2) which was used to immunize rabbits. The anti-FA6S2 rabbit sera revealed a single 43-kDa protein present in Eimeria acervulina, Eimeria tenella, Eimeria maxima and Eimeria falciformis sporulated oocyst antigens. Indirect immunofluorescence and electron microscopy with mAb N3C8B12 localized the putative aspartyl proteinase in the refractile bodies of Eimeria tenella sporozoites.

Superoxide dismutases in Eimeria tenella.

Unsporulated oocysts of Eimeria tenella have high superoxide dismutase (SOD: superoxide:superoxide oxidoreductase, EC 1.15.1.1.) activity and contain several electrophoretically distinct forms of the enzyme, including two forms of Cu/Zn-containing SOD, two forms of Fe-SOD and two forms of Mn-SOD. SOD activity remains high during 12 h of sporulation but diminishes slowly during prolonged sporulation. Oocysts sporulated for 48 h have low levels of superoxide dismutase and contain only one form of the enzyme (Mn-SOD), which was also found in sporozoites. In vitro, sporozoites are oxidant-sensitive and die within minutes of superoxide radical (O2-) generation but SOD/catalase and mannitol protect sporozoites against oxidative damage. These data suggest that E. tenella sporulated oocysts and sporozoites lack soluble cytoplasmic SOD and that this deficiency may contribute to the oxidant sensitivity of the parasite.

Limited heterogeneity between strains of Eimeria tenella isolated from Britain and Bangladesh.

Single-oocyst-derived field strains of Eimeria tenella isolated from Rugby in the United Kingdom (E tenella R) and from Mymensingh and Dhaka in Bangladesh (E tenella M and D, respectively) and a laboratory strain (E tenella, Houghton, H) were compared by isoenzyme electrophoresis, reactivity with antisporozoite monoclonal antibodies and, for some pairs of strains, cross-protection in vivo. The three field strains conformed to one zymodeme with respect to six isoenzymes. For glucose phosphate isomerase (GPI) all field strains were characterised by GPI-9. A panel of six different monoclonal antibodies raised against sporozoites of E tenella H did not discriminate between strains by titration in an immunofluorescence assay against air-dried, acetone fixed sporozoites. In cross-protection experiments involving immunisation and challenge of young chickens, two immunisation schedules were used which, after homologous challenge, provided complete immunity either by the criterion of oocyst output, or by the criterion of weight gain (and more than 94 per cent protection by the criterion of oocyst output). While strain heterogeneity was minimal in the former situation, there was poor cross protection between some strains in the latter case. Under those conditions, heterologous challenge with E tenella M resulted in dysentery and in significantly (P less than 0.05) increased oocyst output and decreased weight gain. The results suggested that E tenella M was immunologically superior to E tenella R and H strains. The results show that a limited degree of immunogenic variability exists between these strains of E tenella and that, unless homologous strain immunity is complete by the criterion of oocyst output, challenge with heterologous strains may result in depressed weight gain.