Tenella Infection In Chickens Research Articles

Expression profile of Toll-like receptors and cytokines in the cecal tonsil of chickens challenged with Eimeria tenella.

Chicken coccidiosis, caused by Eimeria spp., seriously affects the development of the poultry breeding industry. Currently, extensive studies of chicken coccidiosis are mostly focused on acquired immune responses, while information about the innate immune response of chicken coccidiosis is lacking. Toll-like receptor (TLR), the key molecule of the innate immune response, connects innate and adaptive immune responses and induces an immune response against various pathogen infections. Therefore, the quantitative real-time PCR was used to characterize the expression profile of chicken TLRs (chTLRs) and associated cytokines in the cecal tonsil of chickens infected with Eimeria tenella. The results showed that the expression of chTLR1a, chTLR2a, and chTLR5 was significantly upregulated at 3h post-infection, while chTLR1b, chTLR2b, chTLR3, chTLR7, chTLR15 and chTLR21 was significantly downregulated (p < 0.05). In addition, chTLR1a expression rapidly reached the peaked expression at 3h post-infection, while chTLR2b and chTLR15 peaked at 168h post-infection, and chTLR2a expression was highest among chTLRs, peaking at 48h post-infection (p < 0.05). For cytokines, interleukin (IL)-6 and tumor necrosis factor (TNF)-α peaked at 96h post-infection, IL-4 and IL-12 peaked at 144h post-infection, and interferon-γ expression was highest among cytokines at 120h post-infection. In addition, IL-12 and IL-17 were markedly upregulated at 6h post-infection (p < 0.05). These results provide insight into innate immune molecules during E. tenella infection in chickens and suggest that innate immune responses may mediate resistance to chicken coccidiosis.

A rhoptry protein, localizing in the bulb region of rhoptries, could induce protective immunity against Eimeria tenella infection.

Rhoptry organelle proteins (ROPs) secreted by apicomplexan parasites play important roles during parasites invasion and survival in host cells, and are potential vaccine candidates against apicomplexan diseases. Eimeria tenella (E. tenella) is one of the most noteworthy apicomplexan species, which causes hemorrhagic pathologies. Although dozens of putative E. tenella ROP sequences are annotated, most ROP proteins are not well studied. In this study, an E. tenella ROP21 gene was identified and the recombinant EtROP21 protein (rEtROP21) was expressed in Escherichia coli. The developmental expression levels, localization, and protective efficacy against E. tenella infection in chickens were studied. An EtROP21 gene fragment with an open reading frame (ORF) of 981 bp was obtained from the Beijing strain of E. tenella. The rEtROP21 has a molecular weight of approximately 50 kDa and was recognized by rEtROP21-immunized mouse serum. Two specific protein bands, about 43 KDa and 95 KDa in size, were detected in the whole sporozoite proteins using the rEtROP21-immunized chicken serum. RT-qPCR analysis of the E. tenella ROP21 gene (EtROP21) revealed that its mRNA levels were higher in merozoites and sporozoites than in sporulated and unsporulated oocysts. Immunofluorescence and immunoelectron analyses showed that the EtROP21 protein predominantly localizes in the bulb region of rhoptries distributed at anterior, posterior, and perinuclear regions of E. tenella sporozoites. Immunization and challenge experiments revealed that immunizing chickens with rEtROP21 significantly increased their average body weight gain while decreasing mean lesion score and oocyst output (P <0.05). When compared with the challenged control group, the rEtROP21-immunized group was associated with a significantly higher relative weight gain (90.2%) and a greater reduction in oocyst output (67%) (P <0.05). The anticoccidial index of the rEtROP21-immunized group was 163.2. Chicken serum ELISA revealed that the levels of the specific anti- rEtROP21 antibody, IFN-γ, and IL-4 were significantly higher in the rEtROP21-immunized group than in the challenged control group (P <0.05). These results indicate that rEtROP21 can induce a high level of specific immune response and it is a potential candidate for the development of vaccines against E. tenella infection in chickens.

Probiotic Enterococcus faecalis surface-delivering key domain of EtMIC3 proteins: immunoprotective efficacies against Eimeria tenella infection in chickens.

Enterococcus faecalis MDXEF-1 strain was used as host bacteria to deliver surface-displayed key domains BC1 and C4D of the Eimeria tenella microneme 3 (EtMIC3) protein, BC1 fusion to dendritic cell (DC) targeting peptides (DCpep) (DC-BC1) and DC-C4D, respectively. The expression of target proteins in MDXEF-1/BC1-CWA, MDXEF-1/DC-BC1-CWA, MDXEF-1/C4D-CWA, and MDXEF-1/DC-C4D-CWA was verified. The systemic immune responses induced by oral immunization with recombinant E. faecalis were recorded. The immunized chickens were challenged with 4.0 × 104 E. tenella-sporulated oocysts. The immune protections were evaluated by calculating average weight gain and oocyst reduction rate, observing gross and histopathological changes in the cecal tissues, and quantifying levels of inflammatory cytokines in the cecal tissues. The results showed that proteins BC1, C4D, DC-BC1, and DC-C4D were expressed on the surface of E. faecalis, respectively. Oral immunizations with recombinant E. faecalis, especially MDXEF-1/DC-BC1-CWA and MDXEF-1/DC-C4D-CWA, stimulated higher levels of antigen-specific humoral and intestinal mucosal immune responses, higher levels of Th1, Th2, and Th17-type cytokines, and higher proliferation of peripheral blood lymphocytes than phosphate-buffered saline (PBS) and vector control groups (P < 0.01). The groups immunized with four recombinant E. faecalis showed better protective effects against homologous infection than the vector control and infection control groups, and the MDXEF-1/DC-BC1-CWA group displayed the best effects. These results demonstrated that probiotic E. faecalis delivering DCpep fused with the key domain of the EtMIC3 protein could be a potential approach for the prevention of Eimeria infection. IMPORTANCE Avian coccidiosis caused by Eimeria brings huge economic losses to the poultry industry. Although live vaccines and anti-coccidial drugs were used for a long time, Eimeria infection in chicken farms all over the world commonly occurred. The exploration of novel, effective vaccines has become a research hotspot. Eimeria parasites have complex life cycles, and effective antigens are particularly critical to developing anti-coccidial vaccines. Microneme proteins (MICs), secreted from microneme organelles located at the parasite apex, are considered immunodominant antigens. Eimeria tenella microneme 3 (EtMIC3) contains four conserved repeats (MARc1, MARc2, MARc3, and MARc4) and three divergent repeats (MARa, MARb, and MARd), which play a vital role during the Eimeria invasion. Enterococcus faecalis is a native probiotic in animal intestines and can regulate intestinal flora. In this study, BC1 and C4D domains of EtMIC3, BC1 or C4D fusing to dendritic cells targeting peptides, were surface-displyed by E. faecalis, respectively. Oral immunizations were performed to investigate immune protective effects against Eimeria infection.

Transcriptomic, 16S ribosomal ribonucleic acid and network pharmacology analyses shed light on the anticoccidial mechanism of green tea polyphenols against Eimeria tenella infection in Wuliangshan black-boned chickens

BackgroundEimeria tenella is an obligate intracellular parasitic protozoan that invades the chicken cecum and causes coccidiosis, which induces acute lesions and weight loss. Elucidating the anticoccidial mechanism of action of green tea polyphenols could aid the development of anticoccidial drugs and resolve the problem of drug resistance in E. tenella.MethodsWe constructed a model of E. tenella infection in Wuliangshan black-boned chickens, an indigenous breed of Yunnan Province, China, to study the efficacy of green tea polyphenols against the infection. Alterations in gene expression and in the microbial flora in the cecum were analyzed by ribonucleic acid (RNA) sequencing and 16S ribosomal RNA (rRNA) sequencing. Quantitative real-time polymerase chain reaction was used to verify the host gene expression data obtained by RNA sequencing. Network pharmacology and molecular docking were used to clarify the interactions between the component green tea polyphenols and the targeted proteins; potential anticoccidial herbs were also analyzed.ResultsTreatment with the green tea polyphenols led to a reduction in the lesion score and weight loss of the chickens induced by E. tenella infection. The expression of matrix metalloproteinase 7 (MMP7), MMP1, nitric oxide synthase 2 and ephrin type-A receptor 2 was significantly altered in the E. tenella infection plus green tea polyphenol-treated group and in the E. tenella infection group compared with the control group; these genes were also predicted targets of tea polyphenols. Furthermore, the tea polyphenol (-)-epigallocatechin gallate acted on most of the targets, and the molecular docking analysis showed that it has good affinity with interferon induced with helicase C domain 1 protein. 16S ribosomal RNA sequencing showed that the green tea polyphenols had a regulatory effect on changes in the fecal microbiota induced by E. tenella infection. In total, 171 herbs were predicted to act on two or three targets in MMP7, MMP1, nitric oxide synthase 2 and ephrin type-A receptor 2.ConclusionsGreen tea polyphenols can directly or indirectly regulate host gene expression and alter the growth of microbiota. The results presented here shed light on the mechanism of action of green tea polyphenols against E. tenella infection in chickens, and have implications for the development of novel anticoccidial products.Graphical

Comprehensive analysis of gut microbiome and host transcriptome in chickens after Eimeria tenella infection.

Coccidiosis is an intestinal parasitic disease caused by Eimeria protozoa, which endangers the health and growth of animals, and causes huge economic losses to the poultry industry worldwide every year. Studies have shown that poultry gut microbiota plays an important role in preventing the colonization of pathogens and maintaining the health of the host. Coccidia infection also affects host gene expression. However, the underlying potential relationship between gut microbiome and host transcriptome during E. tenella infection in chickens remain unclear. In this study, metagenomic and transcriptome sequencing were applied to identify microbiota and genes in cecal contents and cecal tissues of infected (JS) and control (JC) chickens on day 4.5 postinfection (pi), respectively. First, microbial sequencing results of cecal contents showed that the abundance of Lactobacillus, Roseburia sp. and Faecalibacterium sp decreased significantly after E. tenella infection (P < 0.05), while the abundance of Alistipes and Prevotella pectinovora increased significantly (P < 0.05). Second, transcriptome sequencing results showed that a total of 434 differentially expressed mRNAs were identified, including 196 up-regulated and 238 down-regulated genes. These differentially expressed genes related to inflammation and immunity, such as GAMA, FABP1, F2RL1 and RSAD2, may play an important role in the process of host resistance to coccidia infection. Functional studies showed that the enriched pathways of differentially expressed genes included the TGF-beta signaling pathway and the ErbB signaling pathways. Finally, the integrated analysis of gut microbiome and host transcriptome suggested that Prevotella pectinovora associated with FABP1, Butyricicoccus porcorum and Colidextribacter sp. associated with RSAD2 were involved in the immune response upon E. tenella infection. In conclusion, this study provides valuable information on the microbiota and key immune genes after chicken E. tenella infection, with the aim of providing reference for the impact of coccidia infection on cecal microbiome and host.

Combined oral immunization with probiotics Entercoccus faecalis delivering surface-anchored Eimeria tenella proteins provide protective efficacies against homologous infection in chickens.

Background and ObjectivesAvian coccidiosis is an intestinal parasitic disease exerting a highly negative impact on the global poultry industry. The aim of the present study is to evaluate the immune protective efficacies against Eimeria tenella infection in chickens orally immunized with combined recombinant probiotics Entercoccus faecalis (E. faecalis) delivering surface-anchored E. tenella proteins.MethodsFour kinds of novel probiotics vaccines that surface-expressing four Eimeria tenella (E. tenella) proteins EtAMA1, EtIMP1, EtMIC2 and Et3-1E were produced, respectively. The expression of four target proteins on the surface of recombinant bacteria was detected by Western blot and indirect immunofluorescence assay (IFA). Then the four kinds of recombinant E. faecalis were combined to immunize chickens via oral route in different combinations. The immunizations were performed three times at two-week intervals, and each for three consecutive days. After immunizations, chickens in each immunized group were orally challenged with E. tenella sporulated oocysts. The immune responses and protective efficacies against homologous infection were evaluated.ResultsThe results showed that three or four live recombinant E. faecalis induced effective antigen-specific humoral, intestinal mucosal immune responses, stimulated peripheral T lymphocytes proliferation, and displayed partial protections against homologous challenge as measured by cecal lesions, oocyst shedding, and body weight gain (BWG). Notably, higher levels of protective efficacies were observed when the four recombinant E. faecalis delivering target proteins were combined.ConclusionChickens orally administrated with three or four, especially the four combined recombinant E. faecalis stimulated specific immune responses, which provided anti-coccidial effects. This study offers an idea for future development of novel vaccines based on multi-antigens delivered by probiotic bacteria.

Long noncoding RNA profiling reveals that LncRNA BTN3A2 inhibits the host inflammatory response to Eimeria tenella infection in chickens.

Coccidiosis is a widespread parasitic disease that causes serious economic losses to the poultry industry every year. Long noncoding RNAs (lncRNAs) play important roles in transcriptional regulation and are involved in a variety of diseases and immune responses. However, the lncRNAs associated with Eimeria tenella (E. tenella) resistance have not been identified in chickens. In addition, the expression profiles and functions of lncRNAs during E. tenella infection remain unclear. In the present study, high-throughput sequencing was applied to identify lncRNAs in chicken cecal tissues from control (JC), resistant (JR), and susceptible (JS) groups on day 4.5 post-infection (pi), and functional tests were performed. A total of 564 lncRNAs were differentially expressed, including 263 lncRNAs between the JS and JC groups, 192 between the JR and JS groups, and 109 between the JR and JC groups. Functional analyses indicated that these differentially expressed lncRNAs were involved in pathways related to E. tenella infection, including the NF-kappa B signaling, B cell receptor signaling and natural killer cell-mediated cytotoxicity pathways. Moreover, through cis regulation network analysis of the differentially expressed lncRNAs, we found that a novel lncRNA termed lncRNA BTN3A2 was significantly increased in both cecum tissue and DF-1 cells after coccidia infection or sporozoite stimulation. Functional test data showed that the overexpression of lncRNA BTN3A2 reduced the production of inflammatory cytokines, including IL-6, IL-1β, TNF-α and IL-8, while lncRNA BTN3A2 knockdown promoted the production of these inflammatory cytokines. Taken together, this study identify the differentially expressed lncRNAs during E. tenella infection in chickens for the first time and provide the direct evidence that lncRNA BTN3A2 regulates the host immune response to coccidia infection.

Roles of calpain in the apoptosis of Eimeria tenella host cells at the middle and late developmental stages.

This study investigated the role of calpain in Eimeria tenella-induced host cell apoptosis. Chick embryo cecal epithelial cell culture technology, flow cytometry, enzyme-linked immunosorbent assays, and fluorescence quantitative PCR were used to detect the E. tenella host cell apoptotic rate, Bax and Bid expression levels, and calpain activity. The results demonstrated that Bax, Bid, and calpain levels were upregulated and apoptosis was increased following E. tenella infection at 24-120h. Calpain levels were reduced by pharmacological inhibition of calpain using SJA6017 or by blocking Ca2+ entry into the cell using BAPTA/AM at 24-120h. The mRNA and protein levels of Bax and Bid, the E. tenella infection rate, and the early apoptotic and late apoptotic (necrosis) rates were decreased by using SJA6017 at 24-120h. These results indicated that E. tenella-promoted host cell apoptosis is regulated by calpain via Bid and Bax at 24-120h. Thus, manipulation of calpain levels could be used to manage E. tenella infection in chickens in the middle and late developmental stages.

Dual RNA-seq transcriptome analysis of caecal tissue during primary Eimeria tenella infection in chickens

BackgroundCoccidiosis is an infectious disease with large negative impact on the poultry industry worldwide. It is an enteric infection caused by unicellular Apicomplexan parasites of the genus Eimeria. The present study aimed to gain more knowledge about interactions between parasites and the host immune system during the early asexual replication phase of E. tenella in chicken caeca. For this purpose, chickens were experimentally infected with E. tenella oocysts, sacrificed on days 1–4 and 10 after infection and mRNA from caecal tissues was extracted and sequenced.ResultsDual RNA-seq analysis revealed time-dependent changes in both host and parasite gene expression during the course of the infection. Chicken immune activation was detected from day 3 and onwards with the highest number of differentially expressed immune genes recorded on day 10. Among early (days 3–4) responses up-regulation of genes for matrix metalloproteinases, several chemokines, interferon (IFN)-γ along with IFN-stimulated genes GBP, IRF1 and RSAD2 were noted. Increased expression of genes with immune suppressive/regulatory effects, e.g. IL10, SOCS1, SOCS3, was also observed among early responses. For E. tenella a general up-regulation of genes involved in protein expression and energy metabolism as well as a general down-regulation genes for DNA and RNA processing were observed during the infection. Specific E. tenella genes with altered expression during the experiment include those for proteins in rhoptry and microneme organelles.ConclusionsThe present study provides novel information on both the transcriptional activity of E. tenella during schizogony in ceacal tissue and of the local host responses to parasite invasion during this phase of infection. Results indicate a role for IFN-γ and IFN-stimulated genes in the innate defence against Eimeria replication.

RNA Sequencing Analysis of Chicken Cecum Tissues Following Eimeria tenella Infection in Vivo.

Eimeria tenella (E. tenella) is one of the most frequent and pathogenic species of protozoan parasites of the genus Eimeria that exclusively occupies the cecum, exerting a high economic impact on the poultry industry. To investigate differentially expressed genes (DEGs) in the cecal tissue of Jinghai yellow chickens infected with E. tenella, the molecular response process, and the immune response mechanism during coccidial infection, RNA-seq was used to analyze the cecal tissues of an E. tenella infection group (JS) and an uninfected group (JC) on the seventh day post-infection. The DEGs were screened by functional and pathway enrichment analyses. The results indicated that there were 5477 DEGs (p-value < 0.05) between the JS and the JC groups, of which 2942 were upregulated, and 2535 were downregulated. GO analysis indicated that the top 30 significantly enriched GO terms mainly involved signal transduction, angiogenesis, inflammatory response, and blood vessel development. KEGG analysis revealed that the top significantly enriched signaling pathways included focal adhesion, extracellular matrix–receptor interaction, and peroxisome proliferator-activated receptor. The key DEGs in these pathways included ANGPTL4, ACSL5, VEGFC, MAPK10, and CD44. These genes play an important role in the infection of E. tenella. This study further enhances our understanding of the molecular mechanism of E. tenella infection in chickens.

Molecular characterization and protective efficacy of the microneme 2 protein from Eimeria tenella.

Microneme proteins play an important role in the adherence of apicomplexan parasites to host cells during the invasion process. In this study, the microneme 2 protein from the protozoan parasite Eimeria tenella (EtMIC2) was cloned, characterized, and its protective efficacy as a DNA vaccine investigated. The EtMIC2 gene, which codes for a 35.07 kDa protein in E. tenella sporulated oocysts, was cloned and recombinant EtMIC2 protein (rEtMIC2) was produced in an Escherichia coli expression system. Immunostaining with an anti-rEtMIC2 antibody showed that the EtMIC2 protein mainly localized in the anterior region and membrane of sporozoites, in the cytoplasm of first- and second-generation merozoites, and was strongly expressed during first-stage schizogony. In addition, incubation with specific antibodies against EtMIC2 was found to efficiently reduce the ability of E. tenella sporozoites to invade host cells. Furthermore, animal-challenge experiments demonstrated that immunization with pcDNA3.1(+)-EtMIC2 significantly increased average body weight gain, while decreasing the mean lesion score and oocyst output in chickens. Taken together, these results suggest that EtMIC2 plays an important role in parasite cell invasion and may be a viable candidate for the development of new vaccines against E. tenella infection in chickens.

Evaluation of the protective effect of pVAX-EtMIC3-recombined plasmid against E. tenella in chicken.

Coccidiosis caused by protozoan parasites of the genus Eimeria has a severe economic impact on commercial production worldwide. Micronemes of Eimeria play important roles in invading intestinal cell processes. In this study, the DNA vaccine expressing Eimeria tenella microneme protein 3 (EtMIC3) was constructed to evaluate its immune protective effect against E. tenella infection in chickens. The results demonstrated that chickens immunized with pVAX-EtMIC3 produced strong immune responses in the body, as shown by significant lymphocyte proliferation, cytokine production, and antibody responses. The average body weight gains of chickens in all the vaccinated groups were higher than those of non-vaccinated and challenged groups. In general, oocyst shedding was reduced, and bloody feces and gut lesion scores decreased. In addition, the survival rate of the immunized chickens increased compared to that of the unvaccinated and challenged control chickens. In summary, this study indicated that pVAX-EtMIC3 could induce protective immune effects against coccidiosis and that EtMIC3 is a potential vaccine candidate against coccidiosis.

Molecular characterization and protective efficacy of silent information regulator 2A from Eimeria tenella.

BackgroundSilent information regulator 2 (SIR2) proteins are a family of NAD + -dependent protein deacetylases that are considered potential targets for anti-parasitic agents. In this study, we cloned and characterized SIR2A of the protozoan parasite Eimeria tenella (EtSIR2A) and investigated its protective efficacy as a DNA vaccine.MethodsThe EtSIR2A gene encoding 33.37 kDa protein from E. tenella second-generation merozoites was cloned, and recombinant EtSIR2A protein (rEtSIR2A) was produced in an Escherichia coli expression system. The rEtSIR2A was used to immunize rabbits. Anti-rEtSIR2A antibodies were used to determine the immunolocolization of EtSIR2A in the parasite by immunofluorescence assay (IFA). Transcript and protein expression of EtSIR2A in different development stages of E. tenella were observed by quantitative real-time PCR (qPCR) and western blot (WB) analysis, respectively. The recombinant plasmid pCAGGS-EtSIR2A was constructed and its efficacy against E. tenella infection in chickens was evaluated.ResultsqPCR and WB analysis revealed EtSIR2A expression was developmentally regulated at both the mRNA and protein levels. EtSIR2A mRNA levels were higher in unsporulated oocysts than at other developmental stages, including sporulated oocysts, sporozoites and second-generation merozoites. In contrast, EtSIR2A protein expression levels were highest in second-generation merozoites, moderate in unsporulated oocysts and sporulated oocysts and lowest in sporozoites. Immunostaining with anti-rEtSIR2A antibody indicated that EtSIR2A was mainly located in the cytoplasm of sporozoites and second-generation merozoites, and was strongly expressed during first stage schizogony. Animal-challenge experiments demonstrated that immunization with pCAGGS-EtSIR2A significantly increased average body-weight gain, and decreased mean lesion score and oocyst output in chickens.ConclusionsThese results suggest that EtSIR2A may play an important role in parasite cell survival and may be an effective candidate for the development of new vaccines against E. tenella infection in chickens.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1871-0) contains supplementary material, which is available to authorized users.

Calcium homeostasis in mitochondrion-mediated apoptosis of chick embryo cecal epithelial cells induced by Eimeria tenella infection

In this study, the process of Eimeria tenella-induced apoptosis and the effect of calcium homeostasis were investigated in chick embryo cecal epithelial cells. In particular, we examined cytochrome c release into the cytoplasm, mitochondrial permeability transition pore (MPTP) opening, and changes in [Ca2+]c and apoptosis in host cells. Apoptosis, MPTP opening, cytochrome c release, and [Ca2+]c in host cells increased following infection. This trend was reversed by blocking the increase in [Ca2+]c using BAPTA/AM and EGTA (intra- and extracellular chelators of Ca2+, respectively) and by applying heparin sodium and ryanodine (blockers of the inositol triphosphate and ryanodine receptors of the endoplasmic reticulum, respectively). These results indicate that [Ca2+]c plays a significant role in host cell mitochondrial apoptosis, which is induced via modulation of extracellular Ca2+ levels and endoplasmic reticulum Ca2+ channels. Thus, agents that restore Ca2+ homeostasis may be useful for managing E. tenella infection in chickens.

Effect of Diclazuril on the Bursa of Fabricius Morphology and SIgA Expression in Chickens Infected with Eimeria tenella.

The effects of diclazuril on the bursa of Fabricius (BF) structure and secretory IgA (SIgA) expression in chickens infected with Eimeria tenella were examined. The morphology of the BF was observed by hematoxylin and eosin staining, while ultrastructural changes were monitored by transmission electron microscopy. E. tenella infection caused the BF cell volumes to decrease, irregularly arranged, as well as, enlargement of the intercellular space. Diclazuril treatment alleviated the physical signs of damages associated with E. tenella infection. The SIgA expression in BF was analyzed by immunohistochemistry technique. The SIgA expression increased significantly by 350.4% (P<0.01) after E. tenella infection compared to the normal control group. With the treatment of diclazuril, the SIgA was relatively fewer in the cortex, and the expression level was significantly decreased by 46.7% (P<0.01) compared with the infected and untreated group. In conclusion, E. tenella infection in chickens induced obvious harmful changes in BF morphological structure and stimulated the expression of SIgA in the BF. Diclazuril treatment effectively alleviated the morphological changes. This result demonstrates a method to develop an immunological strategy in coccidiosis control.

Protective immunity against Eimeria tenella infection in chickens following oral immunization with Bacillus subtilis expressing Eimeria tenella 3-1E protein.

The current experiment was conducted to construct recombinant Bacillus subtilis WB600 expressing Eimeria tenella 3-1E protein to investigate the oral immunization protective effects against E. tenella. The merozoite surface antigen 3-1E gene of E. tenella was introduced into the pBS-H1 expression vector with a novel signal peptide sequence. After the electro-transformation, the expression of objective protein in B. subtilis WB600 was detected by Western blot. The results showed that the recombinant B. subtilis strain with the ability of high-level secretion of 3-1E was constructed successfully. Seven-day-old broiler chickens were orally vaccinated with B. subtilis WB600 harboring 3-1E (B.S-pBS-H1-3-1E) or B. subtilis WB600 with empty plasmid (B.S-pBS-H1) 10 days prior to challenge with sporulated E. tenella oocysts. The results showed the recombinant B. subtilis strain with the ability of high-level secretion of 3-1E was constructed successfully. Vaccination with B.S-pBS-H1-3-1E strain significantly increased the anti-coccidial index and reduced cecal lesion scores compared with the positive control group (chickens were challenged with sporulated E. tenella oocysts without oral administration of B.S-pBS-H1-3-1E strain) and B.S-pBS-H1 group. Ceca mucosal sIgA, secretion, and IL-2, IL-12, IFN-γ, and IL-10 level after challenge were greater in the B.S-pBS-H1-3-1E group than in the positive control group. Taken together, these results indicated that B. subtilis WB600 harboring 3-1E protein induces protective immunity against E. tenella.

Protective immunity against Eimeria tenella infection in chickens induced by immunization with a recombinant C-terminal derivative of EtIMP1

Immune mapped protein-1 (IMP1) is a new protective protein in apicomplexan parasites, and exits in Eimeria tenella. Cloning and sequence analysis has predicted the antigen to be a novel membrane protein of apicomplexan parasites. In order to assess the immunogenicity of EtIMP1, a C-terminal derivative of EtIMP1 was expressed in a bacterial host system and was used to immunize chickens. The protective efficacy against a homologous challenge was evaluated by body weight gains, lesion scores and fecal oocyst shedding. The results showed that the subunit vaccine can improve weight gains, reduced cecal pathology and lower oocyst fecal shedding compared with non immunized controls. The results suggested that the C-terminal derivative of EtIMP1 might be considered as a candidate in the development of subunit vaccines against Eimeria infection.

Effect of dinitolmide intercalated into Montmorillonite on E. tenella infection in chickens

To enhance the anti-coccidial effect of dinitolmide and reduce its residual, the dinitolmide/MMT compounds were synthesized by the method of solution intercalation via dinitolmide intercalated into Na + -montmorillonite (Na + -MMT). The structure of compounds was characterized by X-ray diffraction and Fourier transformed infrared. Its anti-coccidial effect was examined by Eimeria tenella infection experiment. One hundred fifty AA broiler chickens were divided into five groups. Chickens were orally inoculated with 5 × 10(4) E. tenella oocysts after dinitolmide was given. Their curative effects were observed. The results showed that intercalated dinitolmide expanded the basal spacing (d 001) of MMT from 12.6 to 15.2 Å. The IR bands of amide group in dinitolmide/MMT were detected at 1,533 cm(-1) which showed that dinitolmide was successfully intercalated into the interlayer spaces of MMT. The dinitolmide/MMT showed higher anti-coccidian oocyst activity compared with dinitolmide (p < 0.05). The dinitolmide/MMT compound can significantly increase body weight gains and reduce bloody diarrhea, lesion score, and oocyst excretion. The anti-coccidia index of dinitolmide/MMT group (165.21) is much higher than dinitolmide group (88.84). The dinitolmide/MMT hybrid systems can be more effective in control of coccidiosis in comparison to dinitolmide.

Anticoccidial activity of traditional Chinese herbal Dichroa febrifuga Lour. extract against Eimeria tenella infection in chickens

The study was conducted on broiler birds to evaluate the anticoccidial efficacy of an extract of Chinese traditional herb Dichroa febrifuga Lour. One hundred broiler birds were assigned to five equal groups. All birds in groups 1-4 were orally infected with 1.5 × 10(4) Eimeira tenella sporulated oocysts and birds in groups 1, 2 and 3 were medicated with 20, 40 mg extract/kg feed and 2 mg diclazuril/kg feed, respectively. The bloody diarrhea, oocyst counts, intestinal lesion scores, and the body weight were recorded to evaluate the anticoccidial efficacy. The results showed that D. febrifuga extract was effective against Eimeria infection; especially 20 mg D. febrifuga extract/kg feed can significantly increase body weight gains and reduce bloody diarrhea, lesion score, and oocyst excretion in comparison to infected-unmedicated control group.

Eimeria tenella: Expression profiling of toll-like receptors and associated cytokines in the cecum of infected day-old and three-week old SPF chickens

Coccidiosis is an economically important protozoan disease worldwide caused by Eimeria parasites. Toll-like receptors (TLRs), a family of highly conserved proteins, are involved in pathogen detection by initiating host responses, and play important roles in the reduction and clearance of pathogens. Little is known about the roles of chicken TLRs during Eimeria tenella infection. We detected the dynamic changes in the expression of TLRs and associated cytokines in the cecum of E. tenella-infected chickens during the early stage of infection. Day-old (Experiment 1) and three-week-old (Experiment 2) chickens were orally gavaged with 10,000 oocysts (30 chickens each experiment), and their cecum intraepithelial lymphocytes were collected at 3, 6, 12, 24, 48, and 72h post-infection (hpi). Expression profiling of TLR1LA, TLR4, TLR5, TLR7, TLR21, and IFN-α, IFN-β, IFN-γ, IL-1β, IL-12 genes were analyzed using quantitative real-time polymerase chain reaction. Almost all TLR transcripts were transiently increased at 3hpi in Experiment 1. In three-week-old chickens, TLR1LA, TLR4, TLR5, TLR7, and TLR21 expression was upregulated at 12hpi, and TLR1LA, TLR5, and TLR21 were highly expressed at 72hpi. In day-old chickens, IFN-α, IFN-β, IFN-γ, IL-1β, and IL-12 expression was significantly upregulated at 3hpi (156.1–1117.1-fold change), in comparison to the different peak level times and relatively small changes for these cytokines in the three-week-old chickens. Our results provide a valuable overview for the expression pattern of TLRs and associated cytokines during the early stage of E. tenella infection in chickens.