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.

Immunomodulatory and ameliorative effects of probiotic in combination with diclazuril on broilers under coccidia infection.

AIM: This study aimed to determine the potential prophylactic efficacy of probiotic individually and/or in combination with anti-coccidial drug on the performance and immunity of broilers under an induced coccidial infection over a 28-day of experimental trial. METHODS: One hundred and eighty 1-day-old Cobb broiler chicks were randomly divided into five groups, included control group (CG), control positive group (CPG), probiotic-treated group (Prob), diclazuril-treated group (Dic), and probiotic + diclazuril-treated group (Prob + Dic). On day 21 of age, all birds, except group CG, were orally inoculated with 1 ml of tap water containing 25,000 Eimeria tenella sporulated oocysts. RESULTS: Our results showed that the probiotic treatment did not influence pre-challenge body weight, feed intake and feed conversion ratio (FCR). During the post-challenge period, chickens in groups probiotic and diclazuril individually and in combination exhibited higher body weight and lower (better) FCR, reduced oocyst shedding (throughout the day four, five, six and seven post-infection), cecal lesions and mortality compared with control positive chickens. Moreover, Compared to CPG group, Prob + Dic group showed increased (p < 0.05) serum levels of interleukin-10 (IL-10) and immunoglobulin M (IgM) and decreased the concentrations of interferon gamma (IFN-γ). On the other hand, individual treatment with probiotic exhibited highest serum levels of IL-10 and IgM, while diclazuril alone increased the blood concentrations of IL-10 and decreased the levels of IFN-γ compared to control positive group; however, there was no significant effect of Prob on IFN-γ, Dic on IgM and all groups on interleukin-17. CONCLUSION: In conclusion, supplementation of probiotic, with and/or without anti-coccidial drug, enhances immunity and inhibits the negative effects of Eimeria infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals the anti-coccidial mechanisms of probiotic in the presence and absence of anti-coccidial drug in preventing the coccidia infection.

In vitro activity of selected natural products against Eimeria tenella sporozoites using reproduction inhibition assay.

Eimeria tenella is the causative agent of cecal coccidiosis in poultry characterized by weight loss, hemorrhagic diarrhea, and high mortality rates. Research into herbal candidates with possible anticoccidial activity has increased lately. As an alternative to animal experiments, an in vitro reproduction inhibition assay (RIA) was previously designed to determine the sensitivity of E. tenella isolates against ionophores. In this study, the RIA was used to test the anticoccidial activity of nutmeg oil, cinnamon oil, and glabridin. The concentration of nutmeg oil used in this study ranged between 1.1 and 139.1 µg/ml. Nutmeg oil exhibited a moderate in vitro inhibitory activity ranging from 35.5 to 49.5%. In contrast, no inhibitory effect was detected when incubating E. tenella sporozoites for 24 h with cinnamon oil at concentrations of 0.3 to 80.5 µg/ml. Glabridin (0.08-41.7 µg/ml) prevented the replication of sporozoites at a rate of 14.1 to 81.7% of inhibition. The calculated minimum concentrations of glabridin needed to inhibit parasite replication by 75%, 50%, and 30% (MIC75, MIC50, and MIC30) were 21.43 µg/ml, 5.28 µg/ml, and 0.96 µg/ml, respectively. Further studies to assess the in vitro efficacy of glabridin were performed by studying mRNA gene expression of stress-induced protein genes (HSP-70, NADPH, and EtPP5) after exposure of E. tenella sporozoites to glabridin at MIC75 for 0.5 h, 1 h, 2 h, and 4 h (a time-dependent experiment). Moreover, a dose-dependent experiment was performed using glabridin at a concentration matching MIC75, MIC50, and MIC30 for 24 h. In the time-dependent experiment, a significant (p < 0.05) increase of expression in NADPH and EtPP5 were detected after 4 h of incubation with glabridin at a concentration of 21.43 µg/ml. The dose-dependent experiment exhibited a gradual increase of expression in all studied genes, which indicates stress imposed on E. tenella sporozoites by glabridin. In our hands, RIA was suitable to assess the anticoccidial activity exhibited by the tested natural products as a precursor to in vivo studies which will help in the identification of novel anticoccidial candidates.

Development of CRISPR-CAS9 based RNA drugs against Eimeria tenella infection.

Parasitic diseases are major trouble in many parts of the world. We consider that if a chemical can break a DNA barcode sequence, it might be used to develop a species-specific anti-parasitic agent. To examine this hypothesis, we constructed sgRNAs that target both the control (5.8S rDNA) and a DNA barcode (ITS) sequence in Eimeria tenella. In vitro experiment showed that Cas9 mRNA combined with sgRNAs could reduce the sporulation percentage of oocysts and the survival rate of sporulated oocysts and sporozoites. Quantitative real-time PCR showed that the DNAs of parasites exposed to Cas9 mRNA and sgRNAs were significantly affected, regardless of whether they were exposed to a combination of two sgRNAs or just a single sgRNA. The DNA sequencing also indicated that the experimental group exposed to two sgRNAs mixed with Cas9-induced deletion of large parts and a single sgRNA mixed with Cas9-induced mutation at sgRNA targeted fragments. In vivo trial, the effect of sgRNA and Cas9 RNA on the pathogenicity of E. tenella in chicken showed less lesion score and oocysts score (P < 0.05) in experimental groups than control groups. The results and concepts presented in this research can lead to discovering novel nucleic acid therapeutic drugs for Eimeriasis and other parasitic infections, which provide insights into the development of species-specific anti-parasitic agents.

Activation of ChTLR15/ChNF-κB-ChNLRP3/ChIL-1ß signaling transduction pathway mediated inflammatory responses to E. tenella infection.

Avian coccidiosis caused by Eimeria leads to severe economic losses in the global poultry industry. Although chicken Toll-like receptor 15 (ChTLR15) was reported to be involved in Eimeria infection, the detailed mechanism underlying its role in the inflammatory response remains to be discovered. The present study demonstrated that the mRNA expression levels of ChTLR15, ChMyD88, ChNF-κB, ChNLRP3, ChCaspase-1, ChIL-18 and ChIL-1ß and the protein levels of ChTLR15 and ChNLRP3 in cecal tissues of Eimeria-infected chickens were significantly elevated at 4, 12, and 24 h compared with those in noninfected control chickens (p < 0.01). Moreover, the mRNA levels of molecules in the ChTLR15/ChNF-κB and ChNLRP3/ChIL-1ß pathways and the protein levels of ChTLR15 and ChNLRP3 in chicken embryo fibroblast cells (DF-1) stimulated by E. tenella sporozoites were consistent with those in Eimeria-infected chickens. Furthermore, overexpression of ChTLR15 in DF1 cells augmented activation of the ChTLR15/ChNF-κB and ChNLRP3/ChIL-1ß pathways when stimulated with E. tenella sporozoites, while knockdown of ChTLR15 in DF1 cells showed inverse effects. Taken together, the present study provides evidence that E. tenella sporozoites specifically activate ChTLR15 and then trigger activation of the ChNLRP3/ChIL-1ß pathway, which partially mediates inflammatory responses to Eimeria infection.

EtMIC3 and its receptors BAG1 and ENDOUL are essential for site-specific invasion of Eimeria tenella in chickens.

Avian coccidian parasites exhibit a high degree of site specificity in different Eimeria species. Although the underlying mechanism is unclear, an increasing body of evidence suggests that site specificity is due to the interaction between microneme proteins (MICs) and their receptors on the surface of target host cells. In this study, the binding ability of E. tenella MICs (EtMICs) to different intestinal tissue was observed by immunofluorescence to identify the key surface molecule on the parasite responsible for the site specificity. Subsequently, we identified the corresponding host-cell receptors by yeast two-hybrid screening and glutathione-S-transferase pull-down experiments and the distribution of these receptors was observed by immunofluorescence in chicken intestinal tissues. Finally, we evaluated the efficacy of receptor antiserum against the infection of E. tenella in chickens. The results showed that EtMIC3 could only bind to the caecum while EtMIC1, EtMIC2, and EtAMA1 did not bind to any other intestinal tissues. Anti-serum to EtMIC3 was able to block the invasion of sporozoites with a blocking rate of 66.3%. The receptors for EtMIC3 were BCL2-associated athanogene 1 (BAG1) and Endonuclease polyU-specific-like (ENDOUL), which were mainly distributed in the caecum. BAG1 and ENDOUL receptor antiserum reduced weight loss and oocyst output following E. tenella infection, showing partial inhibition of E. tenella infection. These data elucidate the mechanism of site specificity for Eimeria infection and reveal a potential therapeutic avenue.

A potential vaccine candidate towards chicken coccidiosis mediated by recombinant Lactobacillus plantarum with surface displayed EtMIC2 protein.

Eimeria tenella (E. tenella) has caused severe economic loss in chicken production, especially after the forbidden use of antibiotics in feed. Considering the drug resistant problem caused by misuse of chemoprophylaxis and live oocyst vaccines can affect the productivity of chickens, also it has the risk to reversion of virulence, the development of efficacious, convenient and safe vaccines is still deeply needed. In this study, the EtMic2 protein of E. tenella was anchored on the surface of Lactobacillus plantarum (L. plantarum) NC8 strain. The newly constructed strain was then used to immunize chickens, followed by E. tenella challenge. The results demonstrated that the recombinant strain could provide efficient protection against E. tenella, shown by increased relative body weight gains, percentages of CD4+ and CD8+ T cells, humoral immune response and inflammatory cytokines. In addition, decreased cecum lesion scores and fecal oocyst shedding were also observed during the experiment. In conclusion, this study proves the possibility to use L. plantarum as a vessel to deliver protective antigen to protect chickens against coccidiosis.

Effects of the PI3K/Akt signaling pathway on the apoptosis of early host cells infected with Eimeria tenella.

This study investigated the role of PI3K/Akt signaling pathway on host cell apoptosis in the early infection of Eimeria tenella. Chicken cecal epithelial cells were treated with apoptosis-inducer Actinomycin D (Act D) or PI3K/Akt signaling pathway inhibitor LY294002 and then infected with E. tenella. Results demonstrated that the E. tenella-infected group had less apoptosis 4-8 h after the infection and more apoptosis 12-20 h after the infection than the control group. At 4-20 h after the infection, the apoptotic/necrotic rate and the Caspase-3 activity in the Act D + E. tenella group were lower (P < 0.01) than those in the Act D-treated group. The p-Akt and NF-κB contents in the E. tenella-infected group were higher (P < 0.01) than those in the control group 4-12 h after the infection. However, the bad content and the Caspase-9/3 activity were lower (P < 0.05) in the E. tenella-infected group than in the control group. Compared with the E. tenella-infected group, the LY294002 + E. tenella group showed decreased p-Akt content and increased apoptotic/necrotic rate, bad content, NF-κB expression, membrane permeability transition pore (MPTP) openness, and Caspase-9/3 activity. Thus, the early development of E. tenella could inhibit host cell apoptosis by downregulating the Caspase-3 activity. Upregulating this activity promoted apoptosis. In addition, activating the PI3K/Akt signaling pathway inhibited the apoptosis of E. tenella host cells in the early infection by reducing the expression of the bad content, limiting the MPTP opening, and decreasing the Caspase-9 and Caspase-3 activities.

Responses to dietary levels of methionine in broilers medicated or vaccinated against coccidia under Eimeria tenella-challenged condition.

BACKGROUND: Coccidiosis is a prevalent problem in chicken production. Dietary addition of coccidiostats and vaccination are two approaches used to suppress coccidia in the practical production. Methionine (Met) is usually the first limiting amino acid that plays important roles in protein metabolism and immune functions in chickens. The present study is aimed to investigate whether increasing dietary Met levels will improve the anticoccidial effects in broilers medicated or vaccinated against coccidia under Eimeria (E.) tenella-challenged condition. Two thousand male Partridge Shank broiler chicks were obtained from a hatchery. After hatch, birds were weighed, color-marked and allocated equally into two anticoccidial treatments, namely medicated and vaccinated groups. Chicks were either fed, from 1 d of age, diets containing coccidiostat (narasin) or diets without the coccidiostat but were inoculated with an anticoccidial vaccine at 3 d of age. At 22 d of age, 1080 chicks among them were randomly allocated evenly into 6 groups under a 2 × 3 treatment with 2 anticoccidial programs and 3 dietary methionine (Met) levels. Chicks medicated or vaccinated against coccidia were fed diets containing 0.45%, 0.56% or 0.68% of Met from 22 to 42 d of age. All chicks were orally introduced with an amount of 5 × 104 sporulated oocysts of E. tenella at 24 d of age. The growth performance, serum anti-oxidative indexes, intestinal morphology, cecal lesion scores, fecal oocyst counts and immune parameters were measured. RESULTS: The results showed increasing dietary Met level from 0.45% to 0.56% and 0.68% improved weight gain and feed conversion of broilers medicated against coccidia. In contrast, higher dietary levels of Met did not improve growth performance of the vaccinated chickens. Higher Met levels helped the medicated chickens resist E. tenella infection, as indicated by improved intestinal morphology and immune functions as well as decreased cecal lesion and fecal oocyst counts. CONCLUSIONS: Anticoccidial vaccination is a better strategy for controlling coccidiosis than feeding narasin, due to not only greater growth performance, but also the lower Met supplementation. Furthermore, higher dietary Met levels improved growth performance of chickens medicated rather than vaccinated against coccidia under E. tenella-challenged condition.

Recombinant lactococcus lactis expressing Eimeria tenella AMA1 protein and its immunological effects against homologous challenge.

Avian coccidiosis leads to severe economic losses for the global poultry industry. Apical membrane antigen 1 (AMA1) of E. tenella (EtAMA1) plays a vital role during invasion of parasites into host cells. In the present study, recombinant live Lactococcus lactis expressing cytoplasmic, secreted and cell wall-anchored EtAMA1 protein were respectively constructed. The three live bacteria were respectively administered orally to SPF chickens (100 µl bacteria containing 5 × 109 CFU per chicken) for three times at 10-day intervals. After immunization, the lymphocyte proliferative function, the percentage of CD4+ and CD8α+ T cells in peripheral blood, and the IgG titers in serum of chickens in each group were respectively measured. The protective effects of live bacteria expressing EtAMA1 protein against E. tenella challenge were evaluated based on body weight gain (BWG), lesion score in cecum, oocyst descrease ratio. The results showed that chickens immunized with three live bacteria, especially the bacteria expressing cell wall-anchored EtAMA1 protein, displayed higher IgG titers and CD4+ T cells proportions, thus provided more immune protective effects against homologous challenge compared with the PBS control group and vector control group (lactococci harboring pTX8048). The oocyst decrease ratio of 33.33% from chickens immunized with lactococci expressing cell wall-anchored EctoAMA1 was observed, which was higher than that of 27.67% and 25.37% from the other two bacteria-immunized groups, respectively. The above results suggested that cell wall-anchored EtAMA1 protein delivered by Lactococcus lactis could stimulate an effective immune responses against Eimeria infection.

Construction of Lactococcus lactis expressing secreted and anchored Eimeria tenella 3-1E protein and comparison of protective immunity against homologous challenge.

Two novel plasmids pTX8048-SP-Δ3-1E and pTX8048-SP-NAΔ3-1E-CWA were constructed. The plasmids were respectively electrotransformed into L. lactis NZ9000 to generate strain of L. lactis/pTX8048-SP-Δ3-1E in which 3-1E protein was expressed in secretion, and L. lactis/pTX8048-SP-NAΔ3-1E-CWA on which 3-1E protein was covalently anchored to the surface of bacteria cells. The expression of target proteins were examined by Western blot. The live lactococci expressing secreted 3-1E protein, anchored 3-1E protein, and cytoplasmic 3-1E protein was administered orally to chickens respectively, and the protective immunity and efficacy were compared by animal experiment. The results showed oral immunization to chickens with recombinant lactococci expressing anchored 3-1E protein elicited high 3-1E-specific serum IgG, increased high proportion of CD4+ and CD8α+ cells in spleen, alleviated average lesion score in cecum, decreased the oocyst output per chicken compared to lactococci expressing cytoplasmic or secreted 3-1E protein. Taken together, these findings indicated the surface anchored Eimeria protein displayed by L. lacits can induce protective immunity and partial protection against homologous infection.

Eimeria tenella: a novel dsRNA virus in E. tenella and its complete genome sequence analysis.

Protozoa double-stranded (ds) RNA viruses have been described in Trichomonas, Giardia, and Leishmania. In this study, dsRNA and virus-like particles (approximately 30 nm in diameter) were discovered in Eimeria tenella sporulated oocysts. The complete genome of this novel dsRNA virus was sequenced using a three-step strategy. The sequencing results showed that the complete genome sequence was 6006 bp containing two open reading frames (ORFs) (2367 bp for ORF1 and 3216 bp for ORF2) with a five-nucleotide overlap (UGA/UG). The predicted ORF1 and ORF2 encoded a putative capsid protein of 788 amino acids (84.922 kDa) and a putative RNA-dependent RNA polymerase (RdRp) protein of 1071 amino acids (118.190 kDa). BLASTp analysis showed that the amino acid sequences for the E. tenella virus shared similarity with the E. brunetti RNA virus, with 29% homology in capsid proteins and 36% in RDRP proteins. The two untranslated regions were 349 bp (5' UTR) and 78 bp (3' UTR). The complete genome sequence of the E. tenella virus resembled characteristics of the Totiviridae family, indicating that this virus was a novel member of Totiviridae. Surprisingly, phylogenetic analysis showed that the E. tenella virus, E. brunetti RNA virus 1, and Mycoviruses were clustered into the genus Victorivirus and separated from the reported protozoa viruses, strongly suggesting a novel Eimeriaviruses subgenus. To the best of our knowledge, this is the first report for the complete genome sequence of the E. tenella virus. Using the nomenclature generally adopted for viruses, this new isolate was named E. tenella RNA virus 1. This study provides a foundation basis for further research on the biological characteristics of Eimeriaviruses.

Ultrastructural effects of acetamizuril on endogenous phases of Eimeria tenella.

To explore the primary stage or site of action of acetamizuril (AZL), a novel triazine anticoccidial compound, the ultrastructural development of Eimeria tenella at different endogenous stages was studied in experimentally infected chickens treated with a single oral dose of 15 mg/kg AZL. As a result of drug action, the differentiations of second-generation schizonts and microgamonts were largely inhibited and merozoites became irregular in shape. Meanwhile, the outer membrane blistering and perinuclear space enlargement were obvious in the second-generation schizonts and microgamonts, which were never observed in the classic triazine anticoccidiosis drug diclazuril-treated E. tenella. The chromatin aggregation, anachromasis, and marginalization were visible in merozoites and microgamonts. Furthermore, the abnormal evagination of microgametes finally resulted in the degeneration of microgamonts and the failure of subsequent fertilization. The most marked micromorphological alteration occurring in the macrogamonts was the WFB2. Even if the fertilization occurred, the formation of oocyst wall became malformed and the zygote proceeded to the obvious degeneration. In addition, mitochondria swelling and cytoplasm vacuolization were discerned in respective intracellular stages, while endoplasmic reticulum and Golgi body swelling was less seen. These alterations may be the causes leading to the final death of E. tenella and also provide some information for further exploring the mechanism of action of AZL at the molecular level.

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.

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

BACKGROUND: The 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. METHODS: In 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. RESULTS: This 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. CONCLUSIONS: An 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.

EtROP38 suppresses apoptosis of host cells infected with Eimeria tenella by inhibition of the p38MAPK pathway.

Coccidiosis is an important parasitic disease that has serious adverse effects on the global poultry industry. The mechanism by which the pathogenic factors of Eimeria tenella damage host cells is unknown. Some kinases from the rhoptry compartment can regulate apoptosis of host cells. This study focused on revealing the role and critical nodes of E. tenella rhoptry protein (EtROP) 38 in controlling the apoptosis of host cells via the P38 mitogen-activated protein kinase (MAPK) signaling pathway. The cells were treated with EtROP38 protein, siRNA p38MAPK, or both. The rate of infection, apoptosis, and the dynamic changes in the expression and activation of key factor genes of the P38MAPK signaling pathway in host cells infected with E. tenella were measured. The results showed that the addition of EtROP38 and/or knockdown of the host cells p38 gene reduced the apoptosis rate of cecal epithelial cells (CECS), decreased the mRNA expressions of p38, p53, c-myc, c-fos, and c-jun and increased the expression of p65, decreased the protein expressions of c-myc, c-fos, and c-jun, decreased the p38 protein phosphorylation level, and increased the p65 protein phosphorylation level in CECS. When E. tenella was inoculated for 4-96 h, the addition of Et ROP38 and/or host cell p38 knockdown both increased the infection rate of host cells, and this effect was more pronounced with the addition of EtROP38 with the host cell p38 knockdown. These observations indicate that E. tenella can inhibits the activation of the p38MAPK signaling pathway in host cells via EtROP38, which suppresses apoptosis in host cells.

Anticoccidial activity of Aloe Vera Leafs' aqueous extract and vaccination against Eimeria tenella: pathological study in broilers.

This study aimed to assess the efficacy of an anticoccidial vaccine and the anticoccidial activity of Aloe vera in broiler chickens infected with Eimeria tenella (E. tenella). A total of 225 healthy, sexless, one-day-old broiler chicks (avian48) from a commercial broiler company were randomized into nine experimental groups of 25 chicks. The groups were as follows: Group 1 (control, vaccinated, non-infected), Group 2 (vaccinated and infected with 5 × 104 sporulated oocysts), Group 3 (vaccinated, infected with 5 × 104 sporulated oocysts, and treated with Aloe vera), Group 4 (infected with 5 × 104 sporulated oocysts and treated with Aloe vera), Group 5 (positive control, infected with 5 × 104 sporulated oocysts), Group 6 (challenged with 5 × 104 sporulated oocysts and then treated with amprolium), Group 7 (treated with amprolium), Group 8 (blank control negative group), and Group 9 (treated with Aloe vera gel).Various parameters were evaluated, including clinical signs, growth performance, oocyst shedding, hematological and immunological parameters, and pathological lesion scoring. The results demonstrated that Aloe vera improved growth performance, reduced oocyst shedding, and decreased caecal lesion scores in E. Tenella-infected broiler chicks. The use of Aloe vera in combination with either amprolium or anticoccidial vaccines provided a potential solution to the issues of drug resistance and drug residues.In conclusion, this study provides valuable insights regarding the control of coccidiosis in broilers. Supplementing the chicken diet with Aloe vera had beneficial effects on the pathogenicity and infectivity of E. tenella, making it a cost-effective alternative as an herbal extract with no adverse side effects for coccidiosis control. These findings suggest that Aloe vera can be considered a potential candidate for inclusion in broiler diets for effective coccidiosis control.

Construction of recombinant SAG22 Bacillus subtilis and its effect on immune protection of coccidia.

Avian coccidiosis causes huge economic losses to the global poultry industry. Vaccine is an important means to prevent and control coccidiosis. In this study, Bacillus subtilis was selected as the expression host strain to express anti Eimeria tenella surface protein SAG22. The synthesized surface protein SAG22 gene fragment of E. tenella was ligated with Escherichia coli-bacillus shuttle vector GJ148 to construct the recombinant vector SAG22-GJ148. And then the recombinant Bacillus strain SAG22-DH61 was obtained by electrotransfer. The results of SDS-PAGE and Western Blot showed that the recombinant protein SAG22 was successfully expressed intracellularly. The immunoprotective effect of recombinant Bacillus strain SAG22-DH61 on broiler chickens was evaluated in 3 identically designed animal experiments. The birds were infected with E. tenella on d 14, 21, and 28, respectively. Each batch of experiments was divided into 4 groups: blank control group (NC), blank control group + infected E. tenella (CON), addition of recombinant SAG22-DH61 strain + infected with E. tenella (SAG22-DH61), addition of recombinant empty vector GJ148-DH61 strain + infected with E. tenella (GJ148-DH61). The animal experiments results showed that the average weight gain of the SAG22-DH61 group was higher than that of the infected control group, and the difference was significant in the d 14 and 28 attack tests (P < 0.05); the oocyst reduction rate of the SAG22-DH61 group was much higher than that of the GJ148-DH61 group (P < 0.05); the intestinal lesion count score of the SAG22-DH61 group was much lower than that of the GJ148-DH61 group (P < 0.05). In addition, the SAG22-DH61 group achieved highly effective coccidia resistance in the d 14 attack test and moderately effective coccidia resistance in both the d 21 and 28 attack tests. In summary, recombinant SAG22 B. subtilis has the potential to become one of the technological reserves in the prevention and control of coccidiosis in chickens in production.

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

Background: 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. Methods: 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. Results: 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. Conclusion: 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.

Botanicals: A promising approach for controlling cecal coccidiosis in poultry.

Avian species have long struggled with the problem of coccidiosis, a disease that affects various parts of the intestine, including the anterior gut, midgut, and hindgut. Among different types of coccidiosis, cecal coccidiosis is particularly dangerous to avian species. Chickens and turkeys are commercial flocks; thus, their parasites have remained critical due to their economic importance. High rates of mortality and morbidity are observed in both chickens and turkeys due to cecal coccidiosis. Coccidiostats and coccidiocidal chemicals have traditionally been added to feed and water to control coccidiosis. However, after the EU banned their use because of issues of resistance and public health, alternative methods are being explored. Vaccines are also being used, but their efficacy and cost-effectiveness remain as challenges. Researchers are attempting to find alternatives, and among the alternatives, botanicals are a promising choice. Botanicals contain multiple active compounds such as phenolics, saponins, terpenes, sulfur compounds, etc., which can kill sporozoites and oocysts and stop the replication of Eimeria. These botanicals are primarily used as anticoccidials due to their antioxidant and immunomodulatory activities. Because of the medicinal properties of botanicals, some commercial products have also been developed. However, further research is needed to confirm their pharmacological effects, mechanisms of action, and methods of concentrated preparation. In this review, an attempt has been made to summarize the plants that have the potential to act as anticoccidials and to explain the mode of action of different compounds found within them.