Inside-out chicken enteroids with leukocyte component as a model to study host-pathogen interactions.

Mammalian three-dimensional (3D) enteroids mirror in vivo intestinal organisation and are powerful tools to investigate intestinal cell biology and host-pathogen interactions. We have developed complex multilobulated 3D chicken enteroids from intestinal embryonic villi and adult crypts. These avian enteroids develop optimally in suspension without the structural support required to produce mammalian enteroids, resulting in an inside-out enteroid conformation with media-facing apical brush borders. Histological and transcriptional analyses show these enteroids comprise of differentiated intestinal epithelial cells bound by cell-cell junctions, and notably, include intraepithelial leukocytes and an inner core of lamina propria leukocytes. The advantageous polarisation of these enteroids has enabled infection of the epithelial apical surface with Salmonella Typhimurium, influenza A virus and Eimeria tenella without the need for micro-injection. We have created a comprehensive model of the chicken intestine which has the potential to explore epithelial and leukocyte interactions and responses in host-pathogen, food science and pharmaceutical research.

Dual RNA-Seq transcriptome analysis of chicken macrophage-like cells (HD11) infected in vitro with Eimeria tenella.

The study aimed to monitor parasite and host gene expression during the early stages of Eimeria tenella infection of chicken cells using dual RNA-Seq analysis. For this, we used chicken macrophage-like cell line HD11 cultures infected in vitro with purified E. tenella sporozoites. Cultures were harvested between 2 and 72 h post-infection and mRNA was extracted and sequenced. Dual RNA-Seq analysis showed clear patterns of altered expression for both parasite and host genes during infection. For example, genes in the chicken immune system showed upregulation early (2­4 h), a strong downregulation of genes across the immune system at 24 h and a repetition of early patterns at 72 h, indicating that invasion by a second generation of parasites was occurring. The observed downregulation may be due to immune self-regulation or to immune evasive mechanisms exerted by E. tenella. Results also suggested pathogen recognition receptors involved in E. tenella innate recognition, MRC2, TLR15 and NLRC5 and showed distinct chemokine and cytokine induction patterns. Moreover, the expression of several functional categories of Eimeria genes, such as rhoptry kinase genes and microneme genes, were also examined, showing distinctive differences which were expressed in sporozoites and merozoites.

Immune response and protective efficacy of recombinant Enterococcus faecalis displaying dendritic cell-targeting peptide fused with Eimeria tenella 3-1E protein.

Avian coccidiosis causes significant economic losses on the global poultry breeding industry. Exploration of new-concept vaccines against coccidiosis has gradually become a research hotspot. In this study, an Enterococcus faecalis strain (MDXEF-1) showing excellent performance isolated from chicken intestinal tract was used as a vector to deliver Eimeria target protein. The plasmid pTX8048-SP-DCpep-NAΔ3-1E-CWA harboring dendritic cell-targeting peptide (DCpep) fusion with Eimeria tenella NAΔ3-1E gene (3-1E protein-coding gene without start codon ATG and terminator codon TAA) was electrotransformed into MDXEF-1 to generate the recombinant bacteria MDXEF-1/pTX8048-SP-DCpep-NAΔ3-1E-CWA in which NAΔ3-1E protein was covalently anchored to the surface of bacteria cells by cell wall anchor (CWA) sequence. The expression of target fusion protein DCpep-NAΔ3-1E-CWA was detected by Western blot. Each chicken was immunized 3 times at 2-wk intervals with live E. faecalis expressing DCpep-NAΔ3-1E fusion protein (DCpep-NAΔ3-1E group), live E. faecalis expressing NAΔ3-1E protein (NAΔ3-1E group), and live E. faecalis containing empty vector only. The 3 immunized groups were then challenged with homologous E. tenella sporulated oocyst after immunizations, and the immune response and protective efficacy in each group were evaluated. The results showed that serum IgG levels, secretory IgA levels in cecal lavage, proportion of CD4+ and CD8α+ cells in peripheral blood, and mRNA expression levels of IL-2 and IFN-γ in the spleen were significantly higher in chickens in the DCpep-NAΔ3-1E group than in chickens of the NAΔ3-1E group (P < 0.05). Oral immunization to chickens with live E. faecalis expressing DCpep-NAΔ3-1E offered more protective efficacy against homologous challenge including significant improved body weight gain, increased oocyst decrease ratio, and reduced average lesion scores in cecum compared with chickens with live E. faecalis expressing NAΔ3-1E protein. These results suggest that recombinant E. faecalis expressing dendritic cell-targeting peptide fusion with E. tenella 3-1E protein could be a potential approach for prevention of Eimeria infection.

Prolonging and enhancing the protective efficacy of the EtMIC3-C-MAR against eimeria tenella through delivered by attenuated salmonella typhimurium.

The microneme adhesive repeats (MAR) of Eimeria tenella microneme protein 3 (EtMIC3) are associated with binding to and invasion of host cells. Adhesion and invasion-related proteins or domains are often strongly immunogenic, immune responses mounted against these factors that play a key role in blocking invasion. In the present study, an oral live vaccine consisting of attenuated Salmonella typhimurium X4550 carrying two MAR domains fragment (St-X4550-MAR) was constructed and its protective efficacies were evaluated. The results showed that St-X4550-MAR was more immunogenic and conferred a higher degree of protection than recombinant MAR polypeptide as reflected by increased body weight, decreased oocyst shedding and lesion scores, increased serum IgG and cecal sIgA antibody production, and increasing levels of interferon-γ and interleukin-10. Thus, MAR domains are highly immunogenic and St-X4550-MAR had moderate activity against E. tenella infection by stimulating humoral, mucosal and cellular immunity. Chickens immunized with our constructed live vaccine provided considerable protections as early as at 10 d post-immunization (ACI: 155.17), and maintained higher protection levels at 20 d post-immunization (ACI: 173.66), and at 30 d post-immunization (ACI: 162.4). While the protective efficacy of chickens immunized with the recombinant MAR peptides showed a decreased trend as the post immunization time prolonging. Thus, using live-attenuated S. typhimurium X4550 as a vaccine expression and delivery system can significantly improve the protective efficacy and duration of protective immunity of MAR of EtMIC3.

Anti-coccidial effect of essential oil blends and vitamin D on broiler chickens vaccinated with purified mixture of coccidian oocyst from Eimeria tenella and Eimeria maxima.

A total of 800 1-day-old Ross 308 male broiler chicks were used in a 5-wk feeding trial to evaluate the effects of commercial feed additive that is composed of blends of essential oil (EO; CRINA Poultry, DSM) and vitamin D3. The birds were randomly assigned to 5 treatments with 10 replicate cages; each stocked with 16 birds and was fed corn-soybean-meal-based basal diet. Dietary treatments included: CON, unvaccinated birds fed basal diet/control; CCO, CON + coccidian oocysts vaccinated birds; EOD1, CCO + Essential oil blends and vitamin D/Prototype 1 (50 µg 25-OH-D3/kg diet, 15 mg CRINA Poultry/kg diet); EOD2, CCO + Prototype 2 (69 µg 25-OH-D3/kg diet, 30 mg CRINA Poultry/kg diet); and EOD3, CCO + Prototype 3 (138 µg 25-OH-D3/kg diet, 50 mg CRINA Poultry/kg diet). On day 7, all birds, except CON, were inoculated with 300 to 500 purified sporulated viable oocysts from Eimeria tenella and Eimeria maxima. As a result of this study, the supplementation of EO blends with vitamin D3 showed trends in improvement (P = 0.075) body weight gain (BWG) compared with CON and CCO during the grower phase (day 7 to 21). An increase (P < 0.05) in BWG and reduction (P < 0.05) in feed conversion ratio (FCR) were observed in birds fed EOD2 diet during day 21 to 35 compared with vaccinated birds without feed additive. The cumulative performance during overall period (day 1 to 35) also showed that BWG was increased and FCR was reduced in EOD2 and EOD3 compared with CON, CCO, and EOD1. Furthermore, the apparent total tract digestibility of dry matter tended (P = 0.06) to increase in EOD2 and EOD3 compared with CON and other treatments. The EOD2 and EOD3 treatments showed increased (P < 0.05) excreta Lactobacillus counts than EOD1. The intestinal lesion score induced by E. tennela in gastro-intestinal tract was lower (P < 0.05) in EOD1, EOD2, and EOD3 treatments compared with CCO. In conclusion, blends of EO and vitamin D3 could be used as anti-coccidial feed additive in broilers with coccidian infection.

Immune response and protective efficacy of Eimeria tenella recombinant refractile body protein, EtSO7, in chickens.

Refractile body protein, SO7, is a highly immunogenic protein which is essentially involved in the early development of Eimeria species infecting the domestic chicken. In the present study, the immune response and protective efficacy of recombinant Eimeria tenella SO7 (rEtSO7) protein was assessed in broiler chickens following homologous oocyst challenge. Broiler chicks were subcutaneously immunized with rEtSO7 antigen adjuvanted with Montanide ISA 71 VG on 7 and 21 days of age and protective efficacy of vaccination was evaluated in terms of body weight gain, lesion score and reduction in oocyst output. The peripheral blood lymphocyte proliferation, serum IgY response, and levels of interferon gamma (IFN-γ), interleukin 2 (IL-2), interleukin 4 (IL-4), tumor growth factor beta (TGF-ß) and nitric oxide (NO) were assessed. The results revealed significant reduction (p < 0.05) in the oocyst output and increased weight gain in immunized birds as compared to unimmunized birds. Significantly increased levels of serum IgY, IFN-γ and proliferation of lymphocytes were evident in rEtSO7 immunized chickens. The results demonstrated that the recombinant protein could effectively elicit the cellular and humoral immune responses in immunized chickens, and provided significant protection against caecal coccidiosis in chickens.

Passive immunization with Eimeria tenella gametocyte antigen 56 (EtGAM56) specific antibodies and active immunization trial with the epitope containing peptide.

Eimeria species cause avian coccidiosis leading to substantial economic losses in the poultry industry. Hence, anticoccidial drugs and vaccines have been used to combat this devitalizing disease. An effective vaccine based on gametocyte recombinant proteins would be very useful in terms of cost, labor and ethics (no animal experimentation). A mouse derived monoclonal antibody against Eimeria tenella gametocyte antigen 56 (EtGAM56) was used to immunize peritoneally E. tenella infected chicken a reduction of oocyst shedding by up to 78% was observed. The epitope recognized by the monoclonal antibody was mapped, recombinant expressed and used to immunize chicken (rEtGAM56N). After rEtGAM56N intramuscular immunization and parasite challenge, clinical parameters like faecal oocyst output, body weight gain, lesion score, feed conversion rate and serum antibody response were assessed to test the efficacy of vaccination against experimental infection with E. tenella. Chicken immunized with rEtGAM56N and challenged with E. tenella oocysts showed a robust antibody response against the rEtGAM56N peptide but no considerable effects on oocyst output and clinical parameters (weight gain, lesion score, feed conversion rate) compared to the mock control group. This study demonstrates the complexity of an effective vaccination. The immunoprotective epitope might be a conformational epitope that was recognized by the monoclonal mouse antibody but only weakly by the antibodies produced against the linear peptide leading to a divergent outcome between the passive and active immunization.

Experimental Toxoplasma gondii and Eimeria tenella co-infection in chickens.

The widespread apicomplexan parasites Toxoplasma gondii (T. gondii) and Eimeria tenella (E. tenella) are important pathogens with high prevalence in poultry. The aim of our study was the investigation of mutual influences in co-infected chickens, focusing on immune response and course of infection. Two separate trials were performed using in total 96 1-day-old chickens, divided into four study groups: group NC (negative control, uninfected), group PC-T (oral or intramuscular infection with T. gondii oocysts (trial 1) or tachyzoites (trial 2), respectively), group PC-E (oral infection with E. tenella (trial 1) or E. tenella and Eimeria acervulina (trial 2)), and group TE (co-infection). T. gondii and Eimeria infections were validated by different parameters, and cytokine expression in the gut and spleen was investigated. T. gondii-specific antibodies were detected earliest 4 days post infection (p.i.) by immunoblot and direct DNA detection was possible in 22.1% of all tissue samples from infected chickens. Eimeria spp. merogony seemed to be enhanced by co-infection with T. gondii, interestingly without marked differences in oocyst excretion between co-infected and Eimeria spp. mono-infected chickens. An increase of messenger RNA (mRNA) expression of Th1- (IFN-γ, IL-12, TNF-α) and Th2-related cytokines (IL-10) mainly in groups PC-E and TE was observed, however, without statistically significant differences between co-infection and single infection with Eimeria. In conclusion, most of the measurable immune response could be attributed to Eimeria infection. To the best of our knowledge, this is the first report on co-infection experiments of T. gondii with Eimeria spp. in chickens.

Screening and characterization of apical membrane antigen 1 interacting proteins in Eimeria tenella.

Avian coccidiosis is a widespread and economically significant disease of poultry. It is an enteric disease caused by several protozoan Eimeria species. Eimeria belongs to the phylum Apicomplexa, which exhibits an unusual mechanism of host cell invasion. During invasion of host cells, the protein apical membrane antigen 1 (AMA1) is essential for invasion of Toxoplasma gondii and Plasmodium. Contrary to the roles of AMA1 during host cell invasion in T. gondii and Plasmodium, the precise functions of Eimeria AMA1 (EtAMA1) are unclear. In order to study the functions of EtAMA1, a yeast two-hybrid cDNA library was constructed from E. tenella sporozoites. The EtAMA1 ectodomain was cloned into the pGBKT7 vector to construct the bait plasmid pGBKT7- EtAMA1. Autoactivation and toxicity of the bait protein in yeast cells were tested by comparison with the pGBKT7 empty vector. Expression of the bait protein was detected by western blots. The bait plasmid pGBKT7-EtAMA1 was used to screen yeast two-hybrid cDNA library from E. tenella sporozoites. After multiple screenings with high-screening-rate medium and exclusion of false-positive plasmids, positive preys were sequenced and analyzed using BLAST. We obtained 14 putative EtAMA1-interacting proteins including E. tenella acidic microneme protein2 (EtMIC2), E. tenella putative cystathionine beta-synthase, E. tenella Eimeria-specific protein, four E. tenella conserved hypothetical proteins (one in the serine/threonine protein kinase family) and seven unknown proteins. Gene Ontology analysis indicated that two known proteins were associated with metabolic process, pyridoxal phosphate binding and protein phosphorylation. Functional analysis indicated EtMIC2 was implicated in parasite motility, migration, recognition and invasion of host cells. The data suggested that EtAMA1 may be important during host cell invasion, but also involved in other biological processes.

Synergy of subgroup J avian leukosis virus and Eimeria tenella to increase pathogenesis in specific-pathogen-free chickens.

To investigate the effects of co-infections of subgroup J avian leukosis virus (ALV-J) and Eimeria tenella on the pathogenesis in specific-pathogen-free (SPF) white leghorn chickens, groups of chickens were infected with ALV-J strain NX0101 at one day of age or with E. tenella at 14 days of age or both. The control group was left uninfected and was mock-inoculated with phosphate buffer saline (PBS). Mortality rates, body weights, cecal lesions, and viremia of infected chickens in each group were evaluated. Immune status was evaluated by measuring several parameters: immune organ weight/body weight index, specific humoral responses to inactivated NDV vaccine and to inoculated E. tenella, proportions of blood CD3+CD4+ and CD3+CD8α+ lymphocytes and transcriptional levels of cytokines in blood and cecal tonsils. The results show that co-infections of ALV-J and E. tenella induced a higher mortality rate and a lower body weight in SPF chickens compared to single-pathogen infection. In co-infected chickens, ALV-J accelerated the disease symptoms induced by E. tenella, and the E. tenella extended the ALV-J viremia. Thymus atrophy, decrease in the humoral response levels to pathogens and the NDV vaccine, modifications in the blood lymphocyte sub-populations and transcriptional cytokine disorders were found in co-infected chickens compared to chickens infected with one pathogen alone and to controls. We underline a synergy between ALV-J and E. tenella that results in increasing pathogenesis in SPF chickens.

Analysis of the function of IL-10 in chickens using specific neutralising antibodies and a sensitive capture ELISA.

In mammals, the inducible cytokine interleukin 10 is a feedback negative regulator of inflammation. To determine the extent to which this function is conserved in birds, recombinant chicken IL-10 was expressed as a secreted human Ig Fc fusion protein (chIL-10-Fc) and used to immunise mice. Five monoclonal antibodies (mAb) which specifically recognise chicken IL-10 were generated and characterised. Two capture ELISA assays were developed which detected native chIL-10 secreted from chicken bone marrow-derived macrophages (chBMMs) stimulated with lipopolysaccharide (LPS). Three of the mAbs detected intracellular IL-10. This was detected in only a subset of the same LPS-stimulated chBMMs. The ELISA assay also detected massive increases in circulating IL-10 in chickens challenged with the coccidial parasite, Eimeria tenella. The same mAbs neutralised the bioactivity of recombinant chIL-10. The role of IL-10 in feedback control was tested in vitro. The neutralising antibodies prevented IL-10-induced inhibition of IFN-γ synthesis by mitogen-activated lymphocytes and increased nitric oxide production in LPS-stimulated chBMMs. The results confirm that IL-10 is an inducible feedback regulator of immune response in chickens, and could be the target for improved vaccine efficacy or breeding strategies.

Evaluation of immune protective efficacies of Eimeria tenella EtMic1 polypeptides with different domain recombination displayed on yeast surface.

In the present study, three different live oral vaccines using the EBY100/pCTCON-2 yeast surface display system with different Eimeria tenella microneme-1 (EtMic1) protein domain recombination were constructed and their protective efficacies against homologous challenge were compared by evaluating the body weight gains, relative growth rate, cecal lesion scores, oocyst output, oocyst decrease ratio, anti-coccidial index, the serum antibody levels and the proliferation ability of blood lymphocytes. The results indicated that all the three constructed live oral vaccines expressing different EtMic1 polypeptides provided excellent protection against homologous challenge by significantly increasing weight gains, reducing cecal lesions, achieving a high ACI, elevating specific antibody response and splenocyte proliferation ability compared with controls. The yeasts displaying the EtMic1 polypeptide-III (expressed TSP-2, TSP-3 and TSP-4 domains) provided better protection against challenge than the yeasts displaying either the EtMic1 polypeptide-I (expressed I-domain, TSP-1 and TSP-2) or polypeptide-II (expressed I-domain and all the five TSP domains) did. Considering the exclusion of antibiotic resistant gene in the system, the strain EBY100 of Saccharomyces cerevisiae may be a better choice for coccidian antigen delivery.

Molecular characterization and functional analysis of subunit 7 of eukaryotic initiation factor 3 from Eimeria tenella.

The initiation of translation in eukaryotic cells is stimulated by proteins known as initiation factors (eIFs). A structurally complex eIF composed of multiple subunits, eIF3 has been shown to have various functions in translation in a variety of eukaryotes. Until now, little is known about eIF3 in Eimeria tenella. Based on a previously identified expressed sequence tag(EST), we cloned the eIF3 subunit 7 gene (EteIF3s7) from E. tenella by rapid amplification of the cDNA ends(RACE). The 2278-bp full-length complementary DNA of EteIF3s7 contained a 1716-bp open reading frame (ORF) that encoded a 571-amino acid (aa) polypeptide. The EteIF3s7 protein contained the subunit 7 domain that is characteristic of members of the eIF3 zeta superfamily. The levels of EteIF3s7 messenger RNA and protein were higher in second generation merozoites than in sporulated oocysts, unsporulated oocysts, or sporozoites, and the EteIF3s7 protein was barely detectable in unsporulated oocysts. Our immunofluorescence analysis showed that the EteIF3s7 protein was uniformly distributed throughout the cytoplasm of sporozoites. After sporozoites were incubated in complete medium, the EteIF3s7 protein localized to the anterior region of the parasite. Following the first schizogenous division, the protein was uniformly dispersed in trophozoites, immature schizonts, and mature schizonts, and the EteIF3s7 protein was observed to be closely associated with the parasitophorous vacuole membrane. An anti-rEteIF3s7 polyclonal antibody inhibited the ability of E. tenella to invade DF-1 cells, which suggested that EteIF3s7 might be involved in host cell invasion and required for the growth of the parasite in the host.

Immunogenicity and protective efficacy of an Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and chicken CD40 ligand.

The CD40 ligand (CD40L) has shown potential as a powerful immunological adjuvant in various studies. Here, the efficacy of a chimeric subunit vaccine, consisting of Eimeria tenella immune mapped protein 1 (EtIMP1) and chicken CD40L, was evaluated against E. tenella infection. The recombinant EtIMP1-CD40L was purified from E. coli over-expressing this protein. Chickens were vaccinated with EtIMP1-CD40L without adjuvant or EtIMP1 with Freund's adjuvant. Immunization of chickens with EtIMP1-CD40L fusion protein resulted in stronger IFN-γ secretion and IgA response than that with only recombinant EtIMP1 with Freund's adjuvant. The clinical effect (cecal lesions, body weights gain, and oocysts shedding) of the EtIMP1-CD40L without adjuvant was also better than that of the EtIMP1 with adjuvant, as evidenced by the difference between the two groups in the oocyst output of E. tenella-challenged chickens. The results suggest that the EtIMP1-CD40L fusion protein can be used as an effective immunogen in the development of subunit vaccines against Eimeria infection.

Different strategies for producing naturally soluble form of common cytokine receptor γ chain.

The common cytokine receptor γ chain (γc) plays an essential role in regulating lymphoid homeostasis. In fact, alteration of this gene causes severe immunodeficiency in humans and animals. Although soluble γc (sγc) was identified in the late 1990s, much remains unknown about its production. This study describes various mechanisms underlying the generation of sγc isoforms in different species. Our data demonstrate that mouse γc and the avian ortholog γc-a did not generate sγc. Moreover, two mouse isoforms, CRA-a and mγc-b, encoded by transcripts lacking a transmembrane region by alternative splicing, did not yield sγc. However, in ducks, sγc was produced from a γc-b transcript lacking a transmembrane region by alternative splicing. In chickens, sγc was produced in normal cells and cell lines by proteolytic shedding of the γc-b isoform containing intron 5, which displayed a relatively high probability of proteolytic cleavage of the ectodomain. This shedding was suppressed by leupeptin, serine and cysteine protease inhibitor. Compared to the chicken ortholog γc-a, expression of γc-b mRNA was differentially regulated according to tissue type, developmental stage, and antigen stimulation. These data demonstrate several mechanisms for producing sγc and suggest a potential role for sγc in avian lymphoid homeostatic responses to environmental antigens.

Eimeria tenella: interleukin 17 contributes to host immunopathology in the gut during experimental infection.

Although IL-17 is a key factor in Th17 lineage host responses and plays critical roles in immunological control of a variety of infectious diseases, the contribution of IL-17 to immune function during Eimeria tenella infection is unknown. In the present study, we used an experimental E. tenella infection model to clarify the role of Th17-associated response in the resulting immune response by quantitative real-time PCR assays. We observed robust production of STAT-3 (the transcription factors), IL-1ß, IL-6 and IL-17 in cecal intraepithelial lymphocytes during the early infection, peaking at 6h p.i. and declining thereafter. The expression of TGF-ß was moderately upregulated and had 2 peaks at 6 and 72h p.i. during the early infection. To further investigate the role of chIL-17 during the infection, we treated the infected chickens with IL-17 and its neutralized antibody. As a result, the reduced fecal oocyst shedding and cecal lesion scores, but enhanced body weight gains were observed in IL-17 neutralized chickens. The results of histopathology showed that the neutrophils recruitment diminished and the parasite burden in IL-17 neutralized chickens decreased. These results may be due to the significant decrease in the production of IL-17, IL-6 and TGF-ß, but enhanced IL-12 and IFN-γ expression in IL-17 neutralized chickens. The converse results were shown in IL-17 treated infected-chickens in which chickens showed increased fecal oocyst shedding, exacerbated lesion scores, and reduced body weight gains. These results suggested that chicken IL-17 might mediate E. tenella - induced immunopathology during the infection.

Immunogenic Eimeria tenella glycosylphosphatidylinositol-anchored surface antigens (SAGs) induce inflammatory responses in avian macrophages.

BACKGROUND: At least 19 glycosylphosphatidylinositol (GPI)-anchored surface antigens (SAGs) are expressed specifically by second-generation merozoites of Eimeria tenella, but the ability of these proteins to stimulate immune responses in the chicken is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Ten SAGs, belonging to two previously defined multigene families (A and B), were expressed as soluble recombinant (r) fusion proteins in E. coli. Chicken macrophages were treated with purified rSAGs and changes in macrophage nitrite production, and in mRNA expression profiles of inducible nitric oxide synthase (iNOS) and of a panel of cytokines were measured. Treatment with rSAGs 4, 5, and 12 induced high levels of macrophage nitric oxide production and IL-1ß mRNA transcription that may contribute to the inflammatory response observed during E. tenella infection. Concomitantly, treatment with rSAGs 4, 5 and 12 suppressed the expression of IL-12 and IFN-γ and elevated that of IL-10, suggesting that during infection these molecules may specifically impair the development of cellular mediated immunity. CONCLUSIONS/SIGNIFICANCE: In summary, some E. tenella SAGs appear to differentially modulate chicken innate and humoral immune responses and those derived from multigene family A (especially rSAG 12) may be more strongly linked with E. tenella pathogenicity associated with the endogenous second generation stages.

Plant expressed EtMIC2 is an effective immunogen in conferring protection against chicken coccidiosis.

Coccidiosis is an economically important disease affecting poultry industry and remains one of the major problems globally. Developing a cost effective sub-unit vaccine may help mitigate loss in the industry. Here, we report expressing one of the microneme proteins, EtMIC2 from Eimeria tenella in tobacco using Agrobacterium-mediated transient expression. The ability of plant expressed recombinant EtMIC2 in eliciting both humoral and cell-mediated immune responses were measured in the immunized birds. The protective efficacy in the vaccinated birds against a homologous challenge was also evaluated. Birds immunized with plant expressed EtMIC2 showed good sero-conversion, reduced oocyst output and increased weight gain when compared to control birds. Our data indicate that use of plant expressed recombinant EtMIC2 in birds was safe and had the potential in imparting partial protection in chickens against homologous challenge.

TRIF is a critical negative regulator of TLR agonist mediated activation of dendritic cells in vivo.

Despite recent advances in developing and licensing adjuvants, there is a great need for more potent formulations to enhance immunogenicity of vaccines. An Eimeria tenella derived antigen (rEA) augments immune responses against several pathogens in animal models and recently was confirmed to be safe for human use. In this study, we have analyzed the molecular mechanisms underlying rEA activity in mice, and confirmed that rEA activates multiple immune cell types, including DCs, macrophages, NK, B, and T cells. The rEA adjuvant also elicits the induction of pleiotropic pro-inflammatory cytokines, responses that completely depend upon the presence of the TLR adaptor protein MyD88. Surprisingly, we also found that the TRIF adaptor protein acts as a potent negative regulator of TLR agonist-triggered immune responses. For example, IL12 production and the induction of co-stimulatory molecule expression by DCs and IFNγ production by NK cells in vivo were significantly increased in rEA-treated TRIF-KO mice. Importantly, however, TRIF suppressive effects were not restricted to rEA-mediated responses, but were apparent in LPS- or ODN2006-activated DCs as well. Taken together, our findings confirm that rEA is a potent adjuvant, triggering robust activation of the innate immune system, in a manner that is augmented by MyD88 and inhibited by TRIF; thereby unveiling the potential complexities of modulating TLR activity to augment vaccine efficacy.

Post-challenge hematological evaluation with virulent strain of Eimeria tenella in broilers immunized with attenuated strain or sporozoite proteins from homologous strain / Avaliação hematológica pós-desafio com cepa virulenta de Eimeria tenella em frangos de corte imunizados com cepa atenuada ou proteínas de esporozoítos de cepa homóloga

Hematological parameters were evaluated in broilers immunized and challenged with Eimeria tenella. Broiler chickens of Hubbard strain, females, coccidian-free, were kept in wire cages and inoculated on the third day. The experiment was designed to include five sorts of treatment with three replicates each. T1 was the negative control group, T2 received 500 attenuated sporulated oocysts by gavage, T3 was the positive control, T4 received 50 µg of sporozoite protein + Quil A vaccine, and T5 received Quil A without sporozoite protein + PBS, the last two through nasal route on days 0, 7, and 21. On the 31st day, all treatments were challenged with homologous virulent strain of E. tenella in the dose of 8.0 × 10(4) oocysts, with the exception of T1. One week later, blood sampling, lesion scores, and cecal oocyst count were carried out. The parasitological parameters showed statistical significance (p < 0.05) and there was no damage to the hematological parameters of birds (p > 0.05) by ANOVA test. The correlations suggest that the blood parameters were impaired by effects of the parasite on tissue, showing levels of hemorrhage and/or hydration.

Foram avaliados os parâmetros hematológicos em frangos de corte imunizados e desafiados com Eimeria tenella. Pintos de corte fêmeas da linhagem Hubbard, livres de coccídios, foram mantidos em baterias metálicas e inoculados no terceiro dia. O experimento foi delineado por cinco tratamentos com três repetições cada, sendo: T1 controle negativo, T2 recebeu 500 oocistos esporulados atenuados via oral, T3 controle positivo, T4 recebeu vacina contendo 50 µg de proteínas de esporozoítos + Quil A e T5 recebeu Quil A + PBS, sendo os dois últimos por via nasal nos dias 0, 7 e 21. No dia 31, todos os tratamentos foram desafiados com cepa virulenta homóloga de E. tenella na dose de 8,0 × 10(4) oocistos, exceto T1. Uma semana depois, foi realizada amostragem de sangue, escore de lesão e contagem de oocistos cecais. Os parâmetros parasitológicos apresentaram significância estatística (p < 0,05), sem que causassem prejuízos aos parâmetros hematológicos das aves (p > 0,05), pelo teste ANOVA. As correlações sugerem que os parâmetros sanguíneos foram afetados pelos efeitos do parasita no tecido, apresentando níveis de hemorragia e/ou hidratação.