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The five epidermal growth factor-like domains (EGF) of Eimeria tenella microneme protein 8 (EtMIC8) (EtMIC8-EGF) plays a vital role in host cell attachment and invasion. These processes require interactions between parasite proteins and receptors on the surface of host cells. In this study, five chicken membrane proteins potentially interacting with EtMIC8-EGF were identified using the GST pull-down assay and mass spectrometry analysis, and only chicken (Gallus gallus) epithelial cell adhesion molecule (EPCAM) could bind to EtMIC8-EGF. EPCAM-specific antibody and recombinant EPCAM protein (rEPCAM) inhibited the EtMIC8-EGF binding to host cells in a concentration-dependent manner. Furthermore, the rEPCAM protein showed a binding activity to sporozoites in vitro, and a significant reduction of E. tenella invasion in DF-1 cells was further observed after pre-incubation of sporozoites with rEPCAM. The specific anti-EPCAM antibody further significantly decreased weight loss, lesion score and oocyst output during E. tenella infection, displaying partial inhibition of E. tenella infection. These results indicate that chicken EPCAM is an important EtMIC8-interacting host protein involved in E. tenella-host cell adhesion and invasion. The findings will contribute to a better understanding of the role of adhesion-associated microneme proteins in E. tenella., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Difficulties of in vitro culture and genetic manipulation of Eimeria tenella have hindered the screening of virulence factors in this parasite. In this study, the E. tenella rhoptry protein 30 (EtROP30) was expressed in Toxoplasma gondii (RH∆Ku80-EtROP30), and its effect on the proliferation and virulence of parasites was investigated. The results revealed that the expression of EtROP30 had no impact on the invasion and egress processes. However, the RH∆Ku80-EtROP30 strain formed larger plaques compared to the RH∆Ku80, indicating that the EtROP30 expression promotes T. gondii proliferation. Furthermore, the RH∆Ku80-EtROP30 strain exhibited greater pathogenicity, resulting in earlier mortality and shorter overall survival time compared to RH∆Ku80. These results imply that EtROP30 expression facilitates parasite intracellular proliferation and virulence in mice, suggesting that EtROP30 might be a candidate virulence factor of E. tenella., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

As drug-resistant strains of Eimeria have emerged and concerns about drug residues in poultry have grown, there is renewed interest in identifying natural alternatives to control coccidiosis. Cedrol, a natural sesquiterpene alcohol, was used in this study to test anticoccidial efficacy in chicks. Both the control and treatment groups were orally challenged with 2 × 10 4 oocysts per chicken. Chicks administered with cedrol had reduced oocyst count, an increase in the relative weight gain rate of chicks, and a decrease in severe swelling of the cecum. Based on the above, ACI was calculated and the cedrol group reached moderate anti-coccidial activity (169.34). In chickens treated with cedrol, there were no changes in serum biochemical parameters, but oxidative stress biomarkers and cytokine levels associated with anticoccidial response were altered. These changes suggest that the administered concentration of cedrol did not have any adverse effects on the chickens while enhancing their antioxidant capacity and immunity, leading to an improved anticoccidial ability. In conclusion, this study shows that the addition of cedrol in poultry production has an anticoccidial effect and successfully improves growth performance during the growth period., Competing Interests: Declaration of Competing Interest We declare that we have no conflicts of interest with respect to the publication of this article., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Avian coccidiosis causes huge economic losses to the poultry industry worldwide and currently lacks effective live vector vaccines. Achieving efficient antigen delivery to mucosa-associated lymphoid tissue (MALT) is critical for improving the effectiveness of vaccines. Here, chicken claudin-3 (CLDN3), a tight junction protein expressed in MALT, was identified as a target, and the C-terminal region of Clostridium perfringens enterotoxin (C-CPE) was proven to bind to chicken CLDN3. Then, a CLDN3-targeting Lactobacillus plantarum NC8-expressing C-CPE surface display system (NC8/GFP-C-CPE) was constructed to successfully express the heterologous protein on the surface of L. plantarum. The colonization level of NC8/GFP-C-CPE was significantly increased compared to the non-targeting strain and could persist in the intestine for at least 72 h. An oral vaccine strain expressing five EGF domains of Eimeria tenella microneme protein 8 (EtMIC8-EGF) (NC8/EtMIC8-EGF-C-CPE) was constructed to evaluate the protective efficacy against E. tenella infection. The results revealed that CLDN3-targeting L. plantarum induced stronger mucosal immunity in gut-associated lymphoid tissues (GALT) as well as humoral responses and conferred better protection in terms of parasite replication and pathology than the non-targeting strain. Overall, we successfully constructed a CLDN3-targeting L. plantarum NC8 surface display system characterized by MALT-targeting, which is an efficient antigen delivery system to confer enhanced protective efficacy in chickens against E. tenella infection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Lactobacillus strains exhibit preferable properties that make them attractive candidates for vaccine delivery systems because of their ability to regulate intestinal mucosal immunity in the body. To date, live Lactobacillus delivery vaccines reported for the defense against Eimeria tenella have been inducer-dependent systems whose applications are significantly limited due to their unattainable induction conditions in vivo. Here, a constitutive expression of Lactobacillus plantarum NC8 surface display system was constructed. Then, this system was used to prepare a live oral vaccine to constitutively express the E. tenella U6L5H2 (EtU6) protein on the NC8 surface and to evaluate its protective efficacy against E. tenella challenge in chickens. The results showed that the heterologous protein (EGFP or EtU6) was successfully expressed on the surface of L. plantarum NC8 without any inducer. The immunoprotection of EtU6 with constitutive expression in L. plantarum NC8 system (NC8/Pc-EtU6) was significantly stronger than that of EtU6 with induced expression of L. plantarum NC8 system (NC8/Pi-EtU6) (ACI: 168.28 vs. 152.74) as evidenced by increased body weight, decreased oocyst output and lesion scores. Furthermore, the constitutive system NC8/Pc-EtU6 produced higher levels of specific cecal SIgA, serum IgG, transcription of cytokines IFN-γ and IL-2, and lymphocyte proliferation than the induced system NC8/Pi-EtU6. These results indicate that, compared to the inducible system, the constitutive surface display system of L. plantarum has the advantages of continuously expressing antigens in vivo and stimulating the host immune system. It could be an ideal platform for vaccine expression. The live vector vaccine for coccidiosis constructed by this constitutive system greatly improves the application potential in chicken production and provides a novel platform for the prevention of coccidiosis in chickens., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Thioredoxin (Trx) is a widespread protein regulator of redox reactions in all organisms. It operates together with NADPH and thioredoxin reductase as a general protein disulfide catalytic system. Recently, Trx has been found to be related to the process by which apicomplexan protozoa invade host cells. In this study, Eimeria tenella thioredoxin (EtTrx1) was identified and its gene structural features, expression levels at different developmental stages, localization in sporozoites, roles in adhesion and invasion, and immunogenicity were investigated. Sequence analysis indicated that EtTrx1 contains a Trx domain with a WCGPC motif in 29-33 aa and a typical Trx fold, and belongs to thioredoxin family. EtTrx1 was detected on the surface of sporozoites using anti-EtTrx1 polyclonal antibodies under non-permeabilized conditions by indirect immunofluorescence assay (IFA) and also in a secretion form. EtTrx1 protein was highly transcribed and expressed in merozoites and sporozoites by quantitative PCR and western blot. The attachment assay showed that the adherence rates of yeast cells expressing EtTrx1 on the surface to host cells were 3.1-fold higher than those of the blank control. Specific anti-EtTrx1 antibodies inhibited the invasion of sporozoites into DF-1 cells. The highest inhibition rate was up to 36.75% compared to the control group. Immunization with recombinant EtTrx1 peptides also showed significant protection against lethal infections in chickens. It could offer moderate protective efficacy (Anticoccidial Index [ACI]: 163.70), induce humoral responses, and be an effective candidate for the development of new vaccines., Competing Interests: Declaration of Competing Interest The authors declare that they have no known personal relationships or competing financial interests that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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

Calcium-binding proteins (CaBPs) containing the specific calcium-binding motif (EF-hand) play a crucial role in important physiological events such as secretion, storage and signal transduction of cells. Recently, CaBPs have been found to be associated with host cell invasions in some parasites. In this study, an Eimeria tenella membrane-associated calcium-binding protein (EtCab) was cloned and its expression at different developmental stages, adhesive functions and host cell invasion in vitro were investigated. The results of the sequence analysis showed that EtCab contains six EF-hand motifs and the HDEL ER-retention signal belonging to the CREC (45 kDa calcium-binding protein, reticulocalbin, ER calcium-binding protein of 55 kDa, and calumenin) family. An indirect immunofluorescence assay (IFA) using specific polyclonal antibodies under permeabilized and nonpermeabilized conditions labeled EtCab on the surface of sporozoites. Quantitative real-time PCR and western blotting indicated that EtCab was highly transcribed and expressed in sporozoites. The attachment assay using a yeast surface display model showed that the adherence rates of EtCab expressed on the surfaces of yeasts to host cells were 2.5-fold greater than the control. Invasion inhibition assays revealed that specific polyclonal antibodies against EtCab significantly reduced the invasion rate of sporozoites on host cells compared to the control group (P < 0.01). These results suggest that EtCab plays an important role in the attachment and invasion of E. tenella to host cells., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Simple Summary: Rhoptry proteins (ROPs) of phylum Apicomplexa parasites are important secretory virulence factors as well as candidate vaccines. However, studies on ROPs of Eimeria tenella are limited. In this study, the coding sequence of E. tenella rhoptry protein 35 (EtROP35) was cloned, and then its localization, expression in parasite, potential role within invasion, and protective efficacy were investigated. Sequence analysis and subcellular localization revealed that EtROP35 is a rhoptry protein of E. tenella. Sporozoite invasion-blocking assay and protective efficacy indicated that EtROP35 might be involved in the parasite invasion process and may be a potential vaccine candidate against E. tenella. Rhoptry proteins (ROPs) of Apicomplexa are crucial secreted virulence factors and sources of vaccine candidates. To date, Eimeria tenella ROPs are not well studied. This study identified and characterized a novel E. tenella ROP (EtROP35), which showed the highest levels among 28 putative ROPs in previous sporozoite and merozoite transcriptomes. Sequence analysis showed that EtROP35 contains an N-terminal secretory signal and a protein kinase domain including eight conserved ROP35-subfamily motifs. Subsequent experiments confirmed that it is a secretory protein. Subcellular localization revealed it localized at the apical end of the sporozoites and merozoites, which was consistent with the ROPs of other Apicomplexan parasites. To further understand the biological meaning of EtROP35, expression levels in different developmental stages and sporozoite invasion-blocking assay were investigated. EtROP35 showed significantly higher levels in sporozoites (6.23-fold) and merozoites (7.00-fold) than sporulated oocysts. Sporozoite invasion-blocking assay revealed that anti-EtROP35 polyclonal antibody significantly reduced the sporozoite invasion rate, suggesting it might participate in host cell invasion and be a viable choice as a vaccine candidate. The immunological protective assays showed that EtROP35 could induce a high level of serum IgY and higher mean body weight gain, and lower cecum lesion score and oocysts excretion than the challenged control group. These data indicated that EtROP35 had good immunogenicity and may be a promising vaccine candidate against E. tenella. [ABSTRACT FROM AUTHOR]