Tenella Infection Research Articles

Histopathologic observations in a coccidiosis model of Eimeria tenella.

Species of the genus Eimeria cause coccidiosis in chickens, resulting in a huge burden to the poultry industry worldwide. Eimeria tenella is one of the most prevalent chicken coccidia in China, and E. tenella infection causes hemorrhagic cecitis. Using an established model of coccidiosis in chickens combined with necropsy, imaging of pathological tissue sections, and other techniques, we evaluated the gross and microscopic lesions of cecal tissue within 15 days after inoculation with sporulated oocysts and described the endogenetic developmental process and relationship between E. tenella infection and enteritis development in chickens. We observed three generations of merogony and gamogony in E. tenella. We observed gross lesions in the cecum from 84 hpi (hours post inoculation) and microscopic lesions from 60 hpi. The lesions in the cecum mainly exhibited hemorrhagic enteritis. Their severity increased with the onset of the second generation of merogony. The lesions began to alleviate by the end of the endogenous stages of E. tenella. We show, for the first time, the complete observation of a series of changes in enteritis caused by 5 × 103 E. tenella oocysts. This study provides reference materials for E. tenella research and pathological diagnosis.

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.

Eimeria tenella rhoptry neck protein 2 plays a key role in the process of invading the host intestinal epithelium

The Apicomplexa parasitic phylum rhoptry neck protein 2 (RON2) plays a key role in the process of invading host cells. Eimeria tenella, an intracellular protozoan shares a similar conserved invasion pattern. However, whether E. tenella RON2 participates in the process of invading the host intestinal epithelium is poorly understood. In this study, the sequence of EtRON2 was analyzed and expressed. The expression of the truncated extracellular N-terminal fragment of EtRON2 (403–700 aa, designated EtRON2403–700) with a molecular mass of 38.3 kDa. EtRON2 in the sporozoite protein was detected at 151.4 kDa by rabbit anti-rEtRON2403–700 antibody. Immunofluorescence results showed that EtRON2 was mainly localized to the nucleus and apex of the E. tenella sporozoite. qPCR results showed that the highest expression level of EtRON2 was detected in sporulated oocysts compared with other developmental stages of E. tenella. In vitro invasion inhibition assays showed that the capacity of sporozoites to invade DF-1 cells was significantly inhibited after pretreatment with the rabbit anti-rEtRON2403–700 antibody. Silencing the EtRON2 gene by RNA interference (RNAi) significantly inhibited EtRON2 expression and significantly reduced the invasion of DF-1 cells by sporozoites. In vivo experiments revealed a significant decrease parasite burden and oocyst outputs in chicks after infection with EtRON2 gene-silenced sporozoites by cloacal inoculation. Recombinant EtRON2403–700 (rEtRON2403–700) immunizes chicks effectively against E. tenella infection by inducing humoral immunity and upregulating IFN-γ and CD8+ T lymphocytes. Furthermore, chicks exhibited increased relative weight gain rates, lower cecum lesion scores, and reduced oocyst outputs during the E. tenella challenge. H&E staining showed that the cecum tissue of chicks immunized with rEtRON2403–700 showed relatively mild histopathological changes. In conclusion, the results of this study demonstrated that EtRON2 plays a key role in E. tenella invasion of the host intestinal epithelium and provides a potential target for vaccines against E. tenella infection.

A Comparison of the Cecal Microbiota between the Infection and Recovery Periods in Chickens with Different Susceptibilities to Eimeria tenella.

To investigate the effect of Eimeria tenella (E. tenella) infection on the cecal microbiota, resistant and susceptible families were screened out based on the coccidiosis resistance evaluation indexes after E. tenella infection. Subsequently, a comparative analysis of cecal microorganisms among control, resistant, and susceptible groups as well as between different periods following the E. tenella challenge was conducted using metagenomic sequencing technology. The results showed that the abundance of opportunistic pathogens, such as Pantoea, Sporomusa, and Pasteurella in the susceptible group and Helicobacter and Sutterella in the resistant group, was significantly higher on day 27 post-inoculation (PI) (the recovery period) than on day 5 PI (the infection period). Additionally, the abundance of Alistipes, Butyricicoccus, and Eubacterium in the susceptible group and Coprococcus, Roseburia, Butyricicoccus, and Lactobacillus in the resistant group showed a significant upward trend during the infection period compared with that in the recovery period. On day 5 PI, the abundance of Faecalibacterium and Lactobacillus was decreased in both the resistant and susceptible groups when compared with that in the control group and was greater in the resistant group than in the susceptible group, while Alistipes in the susceptible group had a relatively higher abundance than that in other groups. A total of 49 biomarker taxa were identified using the linear discriminant analysis (LDA) effect size (LEfSe) method. Of these, the relative abundance of Lactobacillus aviarius, Lactobacillus salivarius, Roseburia, and Ruminococcus gauvreauii was increased in the resistant group, while Bacteroides_sp__AGMB03916, Fusobacterium_mortiferum, Alistipes_sp__An31A, and Alistipes_sp__Marseille_P5061 were enriched in the susceptible group. On day 27 PI, LDA scores identified 43 biomarkers, among which the relative abundance of Elusimicrobium_sp__An273 and Desulfovibrio_sp__An276 was increased in the resistant group, while that of Bacteroides_sp__43_108, Chlamydiia, Chlamydiales, and Sutterella_sp__AM11 39 was augmented in the susceptible group. Our results indicated that E. tenella infection affects the structure of the cecal microbiota during both the challenge and recovery periods. These findings will enhance the understanding of the effects of changes in the cecal microbiota on chickens after coccidia infection and provide a reference for further research on the mechanisms underlying how the intestinal microbiota influence the growth and health of chickens.

Anticoccidial potentials of Azadirachta indica ethosomal nanovesicle in broiler chicks

This study evaluated the efficacy of Azadirachta indica ethosomal nanovesicle against Eimeria tenella infection in broiler chicks. Azadirachta indica ethanolic extract was screened phtochemically and analyzed active components of the extracts using high‑performance liquid chromatography (HPLC). Azadirachta indica ethosomal nanovesicle was synthesized and characterized by zeta potential and scanning electron microscope. Broiler chicks were allocated into seven groups. Control group. The second group administered nanosized ethosomal vesicles (1 mL/kg b.wt.). The third group administered Azadirachta indica nanovesicles (30 mg/kg b.wt.) from 10th day of age. Fourth group was infected with E. tenella at a dose of 1 mL containing 40000 oocyst/ chick at 14th day of age. The fifth group administered Azadirachta indica nanovesicle (30 mg/kg b.wt.) from 10th day of age and infected with E. tenella as fourth group. The sixth group infected with E. tenella as the fourth group and treated with Azadirachta indica nanovesicle (30 mg/kg b.wt. for 4 days after clinical signs appearance. The seventh group infected with E. tenella as the fourth group and treated with diclazuril group (1 mL/4 L of water) for 2 successive days. Coccidiosis significantly decreased body weight, feed intake, reduced glutathione (GSH) level while increased feed conversion ratio, oocyst count, malonaldehyde (MDA) and nitric oxide (NO) serum levels, protein expression of interleukin-1 beta (IL-1β), interleukin 6 (IL-6), BAX and Caspase 3, in cecal tissue and induced cecal tissue injury. However, administration of coccidiosis chicks Azadirachta indica nanovesicle enhanced body weight, and serum GSH. While decreased feed intake, feed conversion ratio, oocyst count, MDA, and NO serum levels, and protein expression of IL-1β, IL-6, BAX, and caspase 3 in cecal tissues and ameliorated cecal tissue damage. This study indicated that, A. indica ethosomal nanovesicle had potent anticoccidial properties.

Efficacy of liver free and Chitosan against Eimeria tenella in chickens

Eimeria spp. are the pathogen that causes coccidiosis, a significant disease that affects intensively reared livestock, especially poultry. Anticoccidial feed additives, chemicals, and ionophores have routinely been employed to reduce Eimeria infections in broiler production. Therefore, the shift to antibiotic-free and organic farming necessitates novel coccidiosis preventive strategies. The present study evaluated the effects of potential feed additives, liver free and chitosan, against Eimeria tenella infection in White Leghorn broiler female chickens. One hundred sixty-five 1-day-old White Leghorn broiler female chicks were divided into 11 groups (15 female chicks per group), including the positive control group (G1), the negative control group (G2), a chitosan-treated group (G3), a chitosan-treated-infected group (G4), the liver free-treated group (G5), the liver free-treated-infected group (G6), the liver free-and-chitosan-treated group (G7), the liver free-and-chitosan-infected group (G8), the therapeutic liver free-and-chitosan-treated-infected group (G9), the sulfaquinoxaline-treated group (G10), and the sulfaquinoxaline-treated-infected group (G11). Chitosan was fed to the chicks in G3 and G4 as a preventative measure at a dose of 250 mg/kg. The G5 and G6 groups received 1.5 mg/kg of Liverfree. The G7 and G8 groups received chitosan and Liverfree. The G10 and G11 groups were administered 2 g/L of sulfaquinoxaline. From the moment the chicks arrived at Foshan University (one-day-old chicks) until the completion of the experiment, all medications were given to them as a preventative measure. G8 did; however, receive chitosan and liver free as therapeutic supplements at 7 dpi. The current study showed that the combination of liver free and chitosan can achieve better prophylactic and therapeutic effects than either alone. In E. tenella challenged chickens, G8 and G9 chickens showed reduced oocyst shedding and lesion score, improved growth performance (body weight, body weight gain, feed intake, feed conversion ratio, and mortality rate), and cecal histology. The current study demonstrates that combining liver free and chitosan has superior preventive and therapeutic benefits than either alone, and they could also be used as alternative anticoccidial agents.

Silymarin effectively prevents and treats Eimeria tenella infection in chicks

Silymarin, a botanical medicine derived from milk thistle seeds and is known to improve chicken growth and gut health when added to the feed. However, its role in the prevention and treatment of chicken coccidiosis remains unclear. This study investigated the efficacy of various doses of silymarin in preventing and treating Eimeria tenella infection in chicks. A total of 180 one-day-old specific pathogen-free chicks were randomized into six groups of 30 chicks each, no treatment (NC group); E. tenella infection (CC group); diclazuril medication during d 14 to 21 and E. tenella infection (DC group); and three groups infected with E. tenella and administered low, medium, or high doses of silymarin during d 12 to 21. All groups except NC were infected with E. tenella on d 14, with indicators observed on d 21. The growth performance was higher in the silymarin treated groups than that in the CC group, and the oocyst count per gram of manure, blood stool, and cecal lesion scores decreased. The medium-dose silymarin group exhibited the best treatment effect. Additionally, the silymarin groups displayed improved histological, morphology, and intestinal barrier integrity. The amounts of proinflammatory factors and harmful bacteria in the cecum were also reduced. Additionally, the activity of serum and cecal antioxidant enzymes, as well as the abundance of beneficial gut microbiota, increased in the cecum. In conclusion, this study demonstrated that silymarin can prevent and treat E. tenella infections. These data provide a scientific and conceptual basis for the development of a botanical dietary supplement from silymarin for the treatment and control of coccidiosis in chicks.

Study on the effects of toosendanin against Eimeria tenella and its impact on the apoptosis of cecal cells in chicks

To investigate the therapeutic effect of toosendanin (TSN) against Eimeria tenella (E. tenella) in chicks. In this experiment, a chick model of artificially induced E. tenella infection was established. The anti-coccidial effect was investigated by treating different doses of TSN. A preliminary mechanism of action was conducted, using cecal cell apoptosis as a starting point. TSN at the concentration of 5 mg/kg BW showed the best effect against E. tenella with the ACI value of 164.35. In addition, TSN reduced pathological damage to cecal tissue, increased the secretion of glycogen and mucus in cecal mucosa, and enhanced the mucosal protective effect. It also elevated the levels of IFN-γ, IL-2, and IgG in serum, and raised the sIgA content in cecal tissue of infected chicks, thereby improving overall immune function. TSN was observed to promote the apoptosis of cecum tissue cells by TUNEL staining analysis. Immunohistochemistry analysis revealed that in TSN-treated groups, the expression of Caspase-3 and Bax was elevated, while the expression of Bcl-2 was reduced. TSN induced apoptosis in host cells by dose-dependently decreasing the Bcl-2/Bax ratio and upregulating Caspase-3 expression. In summary, TSN exhibited significant anticoccidial efficacy by facilitating apoptosis in host cecal cells, with the most pronounced effect observed at a dosage of 5 mg/kg body weight.

Transcriptome analysis reveals that gga-miR-2954 inhibits the inflammatory response against Eimeria tenella infection

Coccidiosis is an important parasitic protozoan disease in poultry farming, causing huge economic losses in the global poultry industry every year. MicroRNAs (miRNAs) are a class of RNA macromolecules that play important roles in the immune response to pathogens. However, the expression profiles and functions of miRNAs during Eimeria tenella (E. tenella) infection in chickens remain mostly uncharacterized. In this study, high-throughput sequencing of cecal tissues of control (JC), resistant (JR), and susceptible (JS) chickens led to the identification of 35 differentially expressed miRNAs among the three groups. Functional enrichment analysis showed that the differentially expressed miRNAs were mainly associated with the TGF-beta, NF-kB, and Jak-STAT signaling pathways. Notably, gga-miR-2954 was found to be significantly upregulated after coccidial infection. Functional analysis showed that gga-miR-2954 inhibited the production of the inflammatory cytokines IL-6, IL-1β, TNF-α, and IL-8 in sporozoite-stimulated DF-1 cells. Mechanistically, we found that gga-miR-2954 targeted the RORC gene and that RORC promoted the inflammatory response in sporozoite-stimulated DF-1 cells. In conclusion, our study was the first to identify differentially expressed miRNAs in chicken cecal tissue during E. tenella infection and found that gga-miR-2954 regulates the host immune response to coccidial infection in chickens by targeting the RORC gene.

Anticoccidial activity of essential oils containing eugenol against Eimeria tenella in broiler chickens

The development of alternative therapies to treat chicken coccidiosis has become a hot topic because of the widespread use of conventional medicines. This study aimed to investigate the effectiveness of eugenol in treating Eimeria tenella infection in broilers. Broiers, at the age of 14 d, were orally infected with sporulated Eimeria tenella oocysts, and then, eugenol essential oil was added to chicken feed at three different dosages (0.1, 0.2 or 0.4 g/kg). The anticoccidial effects of eugenol essential oil were assessed using the anticoccidial index (ACI). As a result, eugenol exhibited a moderate anticoccidial effect, with an ACI of 167.37 at 0.2 g/kg. After eugenol treatment, the expression of occludin in the epithelial cells of the chicken cecum was significantly greater (P < 0.05) than that in the epithelial cells of the nontreated control (IC) group. The proportion of intestinal Lactobacillus_agilli increased. Eugenol therapy dramatically increased the activity of superoxide dismutase. After high-dose treatment, the expression of the proinflammatory factors IL-1β and IL-6 significantly decreased, while the expression of the cytokines IL-4 and IFN-γ significantly increased. The safety of eugenol essential oil was evaluated at the 1, 3 or 6 recommended doses. Overall, no significant differences were detected in the blood tests or serum biochemistry of the chickens between the treatment groups and the control group. As a result, eugenol essential oil can cure chicken coccidiosis by improving the intestinal microbial structure in the chicken cecum and decreasing the cecum's inflammatory reactions, thus strengthening immune function and eventually demonstrating anticoccidial properties.

Curcumin Supplementation Improves Growth Performance and Anticoccidial Index by Improving the Antioxidant Capacity, Inhibiting Inflammatory Responses, and Maintaining Intestinal Barrier Function in Eimeria tenella-Infected Broilers.

This study was conducted to investigate the effects of dietary curcumin supplementation on growth performance, anticoccidial index, antioxidant capacity, intestinal inflammation, and cecum microbiota in broilers infected with Eimeria tenella. A total of 234 one-day-old broilers were categorized into three treatments, with six replicates per treatment containing 13 broilers each. The three treatments included the control group, Eimeria tenella group, and Eimeria tenella + curcumin (200 mg/kg) group. The feeding trial lasted for 42 days, during which the broilers were orally administered with 0.9% saline or 5 × 104Eimeria tenella oocysts on day 14 of the study. On day 17 and day 21, one bird per replicate was selected for slaughtering. Results indicated an increased survival rate and anticoccidial index and improved productive performance in coccidia-infected broilers with curcumin supplementation. Furthermore, curcumin enhanced the serum antioxidant capacity in Eimeria tenella-infected broilers, evidenced by increased serum catalase activity (3d, 7d), as well as decreased malondialdehyde level (3d, 7d) and nitric oxide synthase activity (7d) (p < 0.05). Curcumin also improved intestinal inflammation and barrier function, evidenced by the downregulation of interleukin (IL)-1β (3d, 7d), TNF-alpha (TNF-α) (3d, 7d), and IL-2 (7d) and the up-regulated mRNA levels of claudin-1 (7d), zonula occludens (ZO-1; 3d, 7d), and occludin (3d, 7d) in the ceca of infected broilers (p < 0.05). Eimeria tenella infection significantly disrupted cecum microbial balance, but curcumin did not alleviate cecum microbial disorder in broilers infected with Eimeria tenella. Collectively, curcumin supplementation enhanced growth performance and anticoccidial index in Eimeria tenella-infected broilers via improving antioxidant ability and cecum inflammation without affecting cecum microbiota.

Global profiling of protein S-palmitoylation in the second-generation merozoites of Eimeria tenella.

The intracellular protozoan Eimeria tenella is responsible for avian coccidiosis which is characterized by host intestinal damage. During developmental cycle, E. tenella undergoes versatile transitional stages such as oocyst, sporozoites, merozoites, and gametocytes. These developmental transitions involve changes in cell shape and cell size requiring cytoskeletal remodeling and changes in membrane proteins, which may require transcriptional and translational regulations as well as post-translational modification of proteins. Palmitoylation is a post-translational modification (PTM) of protein that orchestrates protein targeting, folding, stability, regulated enzymatic activity and even epigenetic regulation of gene expression. Previous research revealed that protein palmitoylation play essential role in Toxoplasma gondii, Trypanosoma cruzi, Trichomonas vaginalis, and several Plasmodium parasites. Until now, there is little information on the enzymes related to palmitoylation and role of protein acylation or palmitoylation in E. tenella. Therefore, palmitome of the second-generation merozoite of E. tenella was investigated. We identified a total of 2569 palmitoyl-sites that were assigned to 2145 palmitoyl-peptides belonging to 1561 protein-groups that participated in biological processes including parasite morphology, motility and host cell invasion. In addition, RNA biosynthesis, protein biosynthesis, folding, proteasome-ubiquitin degradation, and enzymes involved in PTMs, carbohydrate metabolism, glycan biosynthesis, and mitochondrial respiratory chain as well as vesicle trafficking were identified. The study allowed us to decipher the broad influence of palmitoylation in E. tenella biology, and its potential roles in the pathobiology of E. tenella infection. Raw data are publicly available at iProX with the dataset identifier PXD045061.

Prophylactic and Therapeutic Efficacy of Ultrasonicated Rosmarinus officinalis Ethanolic Extract and its Chitosan-Loaded Nanoparticles Against Eimeria tenella Infected Broiler Chickens

PurposeThe in vivo efficacy of ultrasonicated Rosmarinus officinalis ethanolic extract (UROEE) and its chitosan-loaded nanoparticles (UROEE-CsNPs) was investigated as a dietary prophylactic agent and as a therapeutic treatment against Eimeria tenella infected broiler chickens.MethodsChickens were infected with 4 × 104E. tenella oocysts at 21 days old for primary infection and with 8 × 104 oocysts at 35 days old for secondary infection. Eleven experimental groups were conducted. Dietary addition of 100 mg/kg UROEE and 20 mg/kg for CsNPs as well as UROEE-CsNPs were included for prophylactic groups from day 1 to 42. The same doses were used for therapeutic treatment groups for 5 constitutive days. Oocyst output in feces was counted. Histopathological and immunohistochemical studies were conducted. Gene expression of pro-inflammatory cytokines as IFN-γ, IL-1β and IL-6 as well as anti-inflammatory cytokines as IL-10 and TGF-β4 was analyzed using semi-quantitative reverse transcriptase-PCR.ResultsThe results showed an efficacy of UROEE, CsNPs and UROEE-CsNPs in reduction of oocyst excretion and improving the cecal tissue architecture. CD4+ and CD8+ T lymphocytes protein expression were reduced. E. tenella infection lead to upregulation of pro-inflammatory cytokines as IFN-γ, IL-1β, IL-6 and anti-inflammatory cytokines as TGF-β4 following primary infection, while their expression was downregulated following secondary infection.ConclusionThe dietary prophylactic additives and therapeutic treatments with UROEE, CsNPs and UROEE-CsNPs could decrease the inflammatory response to E. tenella as indicated by oocyst output reduction, histopathological improvements, CD4+ and CD8+ T cells protein expression reduction as well as reducing mRNA expression levels of the tested cytokines following primary and secondary infections. Consequently, these results will help to develop better-combating strategies for the control and prevention of coccidiosis on poultry farms as a dietary prophylactic agent or as a therapeutic treatment.Graphical

Recombinant production of SAG1 fused with xylanase in Pichia pastoris induced higher protective immunity against Eimeria tenella infection in chicken.

Chicken coccidiosis is an intestinal disease caused by the parasite Eimeria, which severely damages the growth of chickens and causes significant economic losses in the poultry industry. Improvement of the immune protective effect of antigens to develop high efficiency subunit vaccines is one of the hotspots in coccidiosis research. Sporozoite-specific surface antigen 1 (SAG1) of Eimeria tenella (E. tenella) is a well-known protective antigen and is one of the main target antigens for the development of subunit, DNA and vector vaccines. However, the production and immunoprotective effects of SAG1 need to be further improved. Here, we report that both SAG1 from E. tenella and its fusion protein with the xylanase XynCDBFV-SAG1 are recombinant expressed and produced in Pichia pastoris (P. pastoris). The substantial expression quantity of fusion protein XynCDBFV-SAG1 is achieved through fermentation in a 15-L bioreactor, reaching up to about 2 g/L. Moreover, chickens immunized with the fusion protein induced higher protective immunity as evidenced by a significant reduction in the shedding of oocysts after E. tenella challenge infection compared with immunized with recombinant SAG1. Our results indicate that the xylanase enhances the immunogenicity of subunit antigens and has the potential for developing novel molecular adjuvants. The high expression level of fusion protein XynCDBFV-SAG1 in P. pastoris holds promise for the development of effective recombinant anti-coccidial subunit vaccine.

Expression of Toll-like receptors and host defence peptides in the cecum of chicken challenged with Eimeria tenella.

Chicken coccidiosis, caused by Eimeria protozoa, affects poultry farming. Toll-like receptors (TLRs) and host defence peptides (HDPs) help host innate immune responses to eliminate invading pathogens, but their roles in Eimeria tenella infection remain poorly understood. Herein, 14-day-old chickens were treated orally with 50,000 E. tenella oocysts and the cecum was dissected at different timepoints. mRNA expression of 10 chicken TLRs (chTLRs) and five HDPs was measured by quantitative real-time PCR. chTLR7 and chTLR15 were upregulated significantly at 3 h post-infection while other chTLRs were downregulated (p < .05). chTLR1a, chTLR1b, chTLR2b and chTLR4 peaked at 36 h post-infection, chTLR3, chTLR5 and chTLR15 peaked at 72 h post-infection and chTLR21 expression was highest among chTLRs, peaking at 48 h post-infection (p < 0.05). For HDPs, cathelicidin (CATH) 1 to 3 and B1 peaked at 48 h post-infection, liver-expressed antimicrobial peptide 2 peaked at 96 h post-infection, and CATH 2 expression was highest among HDPs. CATH2 and CATH3 were markedly upregulated at 3 h post-infection (p < .05). The results provide insight into innate immune molecules during E. tenella infection in chicken, and indicate that innate immune responses may mediate resistance to chicken coccidiosis.

Host protein EPCAM interacting with EtMIC8-EGF is essential for attachment and invasion of Eimeria tenella in chickens

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.

A novel avian intestinal epithelial cell line: its characterization and exploration as an in vitro infection culture model for Eimeria species

BackgroundThe gastrointestinal epithelium plays an important role in directing recognition by the immune system, and epithelial cells provide the host's front line of defense against microorganisms. However, it is difficult to cultivate avian intestinal epithelial cells in vitro for lengthy periods, and the lack of available cell lines limits the research on avian intestinal diseases and nutritional regulation. Chicken coccidiosis is a serious intestinal disease that causes significant economic losses in the poultry industry. In vitro, some cell line models are beneficial for the development of Eimeria species; however, only partial reproduction can be achieved. Therefore, we sought to develop a new model with both the natural host and epithelial cell phenotypes.MethodsIn this study, we use the SV40 large T antigen (SV40T) gene to generate an immortalized cell line. Single-cell screening technology was used to sort positive cell clusters with epithelial characteristics for passage. Polymerase chain reaction (PCR) identification, immunofluorescence detection, and bulk RNA sequencing analysis and validation were used to check the expression of epithelial cell markers and characterize the avian intestinal epithelial cell line (AIEC). AIECs were infected with sporozoites, and their ability to support the in vitro endogenous development of Eimeria tenella was assessed.ResultsThis novel AIEC consistently expressed intestinal epithelial markers. Transcriptome assays revealed the upregulation of genes associated with proliferation and downregulation of genes associated with apoptosis. We sought to compare E. tenella infection between an existing fibroblast cell line (DF-1) and several passages of AIEC and found that the invasion efficiency was significantly increased relative to that of chicken fibroblast cell lines.ConclusionsAn AIEC will serve as a better in vitro research model, especially in the study of Eimeria species development and the mechanisms of parasite–host interactions. Using AIEC helps us understand the involvement of intestinal epithelial cells in the digestive tract and the immune defense of the chickens, which will contribute to the epithelial innate defense against microbial infection in the gastrointestinal tract.Graphical

CRISPR-Cas9-based method for isolating microgametes of Eimeria tenella

Eimeria tenella infections are known to cause severe caecal damage and death of the infected chicken. Gamogony is an essential stage in E. tenella life cycle and in the establishment of coccidiosis. Prior research had extensively explored isolation and separation of the parasite gametes - microgamete (male) and macrogamete (female). However, there is little information on the efficient, highly purified and distinctly separated male and female gametes. In this study, we generated a genome editing line expressing mCherry fluorescent protein fused with GCS1 protein in E. tenella by using Toxoplasma gondii CRISPR-Cas9 system, flow cytometry and fluorescence microscopy. This allowed precise separation of E. tenella male and female gametes in the transgenic parasite population. The separation of male and female gametes would not only build on our understanding of E. tenella transmission, but it would also facilitate development of gametocidal compounds as drug targets for E. tenella infection.

Research Note: Preliminary functional analysis of EGF-like domains of Eimeria tenella microneme protein 7 identified in sporozoites and merozoites

Eimeria tenella, an obligate intracellular apicomplexan parasite, is the major causative agent of chicken coccidiosis. Some epidermal growth factor (EGF)-like domain-containing proteins of other members of apicomplexan parasites have been reported to contribute to parasite survival. To date, however, EGF-like domain-containing proteins of E. tenella are not well studied. In this study, a gene fragment that encodes 4 EGF-like domains of E. tenella microneme protein 7 (EGF-EtMIC7) was amplified and expressed using an Escherichia coli expression system. Following generation of polyclonal antibodies that recognize recombinant EGF-EtMIC7 (rEGF-EtMIC7), the expression of EtMIC7 in sporozoites and merozoites was examined. Moreover, its roles in cellular regulation were investigated. The native EtMIC7 in E. tenella sporozoites and merozoites was detected by using Western blot and indirect immunofluorescence assays. rEGF-EtMIC7 could activate Akt, whereas blockade of EGF receptor (EGFR) failed to induce Akt phosphorylation. Compared with the control group, LMH cells treated with rEGF-EtMIC7 showed increased cell proliferation and expressed higher levels of B cell leukemia/lymphoma 2 (BCL-2). These findings contribute to the better understanding of parasite-host interactions at the molecular level during E. tenella infection.

Quantitative phosphoproteomic analysis of chicken DF-1 cells infected with Eimeria tenella, using tandem mass tag (TMT) and parallel reaction monitoring (PRM) mass spectrometry.

Eimeria tenella is an obligate intracellular parasite which causes great harm to the poultry breeding industry. Protein phosphorylation plays a vital role in host cell-E. tenella interactions. However, no comprehensive phosphoproteomic analyses of host cells at various phases of E. tenella infection have been published. In this study, quantitative phosphoproteomic analysis of chicken embryo DF-1 fibroblasts that were uninfected (UI) or infected with E. tenella for 6h (PI6, the early invasion phase) or 36h (PI36, the trophozoite development phase) was conducted. A total of 10,122 phosphopeptides matched to 3,398 host cell phosphoproteins were identified and 13,437 phosphorylation sites were identified. Of these, 491, 1,253, and 275 differentially expressed phosphorylated proteins were identified in the PI6/UI, PI36/UI, and PI36/PI6 comparisons, respectively. KEGG pathway enrichment analysis showed that E. tenella modulated host cell processes through phosphorylation, including focal adhesion, regulation of the actin cytoskeleton, and FoxO signaling to support its early invasion phase, and modulating adherens junctions and the ErbB signaling pathway to favor its trophozoite development. These results enrich the data on the interaction between E. tenella and host cells and facilitate a better understanding of the molecular mechanisms underlying host-parasite relationships.