mRNA 5-methylcytosine in Eimeria tenella oocysts: An abundant post-transcriptional modification associated with broad-ranging biological processes.

Eimeria tenella is the major causative agent of chicken coccidiosis. 5-Methylcytosine (m5C) is a type of RNA chemical modifications reported to regulate diverse biological processes. However, the distribution and biological functions of m5C in E. tenella mRNAs are yet to be known. Herein, we report transcriptome-wide profiling of mRNA m5C in E. tenella by employing m5C RNA immunoprecipitation followed by a deep-sequencing approach (m5C-RIP-seq). Our data showed that m5C peaks were distributed across the whole mRNA body. Compared with unsporulated oocysts, there were 2813 hypermethylated and 1850 hypomethylated m5C peaks in sporulated oocysts. Generally, a positive correlation between m5C modification and gene expression levels was observed. The mRNA sequencing (RNA-seq) and m5C-RIP-seq data were consistent with the results of the quantitative reverse transcription PCR (RT-qPCR) and methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR), respectively. Gene Ontology (GO) and pathway enrichment analysis predicated diverse biological functions and pathways, including microtubule motor activity, helicase activity, cGMP-PKG signaling pathway, aminoacyl-tRNA biosynthesis, glycolysis/gluconeogenesis, and spliceosome. Meanwhile, stage-specific gene expression signatures of m5C-related regulators were observed. Altogether, our findings reveal the transcriptional significance of m5C modification in E. tenella oocysts, providing resources and clues for further in-depth research.

Anticoccidial activity of the secondary metabolites in alpine plants frequently ingested by wild Japanese rock ptarmigans.

The Japanese rock ptarmigan (Lagopus muta japonica) is an herbivorous species of partridges that inhabits only alpine zones. Alpine plants are their main source of food. These alpine plants contain toxic compounds to deter herbivores from consuming them. A previous analysis of the alpine plants frequently consumed by Japanese rock ptarmigans revealed the presence of a unique mixture of secondary metabolites and a novel compound. Additionally, wild Japanese rock ptarmigans are often infected by two species of Eimeria parasites. When these parasites were experimentally administered to Svalbard rock ptarmigans (Lagopus muta hyperborean), which do not feed on alpine plants, the birds exhibited symptoms, such as diarrhea and depression, and in some cases, they died. Although little is known about the pathogenesis of these parasites in wild Japanese rock ptarmigans, it was hypothesized that compounds found in alpine plants, their main food source, may reduce the pathogenicity of Eimeria parasites. In the present study, we evaluated the anticoccidial activity of the compounds derived from alpine plants in vitro using Eimeria tenella, which infects chickens belonging to the same pheasant family, as an experimental model. Twenty-seven natural components were extracted from eight alpine plants. The natural components were added to E. tenella sporozoites and incubated for 24 h to evaluate their direct effect. Additionally, Madin-Darby bovine kidney cells were incubated with sporozoites and natural components for 24 h to evaluate the inhibitory effect of the components on sporozoite cell invasion. Six compounds from four alpine plants decreased sporozoite viability by up to 88.3%, and two compounds inhibited sporozoite invasion into the cells. Although further studies are needed to evaluate the effects of these components against Eimeria infections in vivo, our findings suggest that these alpine plants may reduce the degree of infection by decreasing the number of sporozoites in the intestinal tract.

Drug resistance and genetic characteristics of one Eimeria tenella isolate from Xiantao, Hubei Province, China.

Chicken coccidiosis causes retarded growth and low production performance in poultry, resulting in huge economic losses to the poultry industry. In order to prevent and control chicken coccidiosis, great efforts have been made to develop new drugs and vaccines, which require pure isolates of Eimeria spp. In this study, we obtained the Eimeira tenella Xiantao isolate by single oocyst isolation technology and compared its genome with the reference genome GCF_000499545.2_ETH001 of the Houghton strain. The results of the comparative genomic analysis indicated that the genome of this isolate contained 46,888 single nucleotide polymorphisms (SNPs). There were 15,107 small insertion and deletion variations (indels), 1693 structural variations (SV), and 3578 copy number variations (CNV). In addition, 64 broilers were used to determine the resistance profile of Xiantao strain. Drug susceptibility testing revealed that this isolate was completely resistant to monensin, diclazuril, halofuginone, sulfachlorpyrazine sodium, and toltrazuril, but sensitive to decoquinate. These data improve our understanding of drug resistance in avian coccidia.

Anticoccidial activity of a botanical natural product based on eucalyptus, apigenin and eugenol against Eimeria tenella in broiler chickens.

BACKGROUND: Chicken coccidiosis is an intracellular parasitic disease that presents major challenges to the development of the commercial poultry industry. Perennial drug selective pressure has led to the multi-drug resistance of chicken coccidia, which makes the prevention and control of chicken coccidiosis extremely difficult. In recent years, natural plant products have attracted the attention of researchers due to their inherent advantages, such as the absence of veterinary drug residues. The development of these natural products provides a new direction for the prevention and treatment of chicken coccidiosis. METHODS: The anticoccidial effect of a natural plant product combination formulation (eucalyptus oil + apigenin + eugenol essential oil) was tested against Eimeria tenella in broilers. To search for the optimal concentration of the combination formulation, we screened 120 broilers in a chicken cage trial in which 100 broilers were infected with 5 × 104 sporulated Eimeria tenella oocysts; broilers receiving a decoquinate solution was set up as a chemical control. The optimal anticoccidial concentration was determined by calculating the anticoccidial index (ACI), and the suitable concentration was used as the recommended dose for a series of safety dose assessment tests, such as feed conversion ratio (FCR), hematological indices and serum biochemical indices, as well as liver and kidney sections, at onefold (low dose), threefold (medium dose) and sixfold (high dose) the recommended dose (RD). RESULTS: The results showed that this combination formulation of three plant natural products had a better anticoccidial effect than formulations containing two plant natural products or a single one, with an ACI of 169.3. The dose gradient anticoccidial test revealed that the high-dose formulation group had a better anticoccidial effect (ACI = 169.2) than the medium- and low-dose groups. The safety evaluation test showed that concentrations of the formulation at one-, three- and sixfold the RD were non-toxic to Arbor Acres broilers, indicating the high safety of the combination formulation. CONCLUSIONS: The combination formulation showed not only a moderate anticoccidial effect but also had a high safety profile for broilers. The results of this study indicate a new alternative for the prevention and control of coccidiosis in broilers.

Traditional Chinese medicine formulation ChangQing compound has significant therapeutic effects on chickens infected with Eimeria tenella.

Coccidiosis poses a significant challenge to the poultry industry. However, the excessive and improper use of anticoccidial drugs and vaccines has led to resistance and food safety concerns. Consequently, traditional Chinese herbs have garnered attention as a potentially safer and more effective alternative. ChangQing compound derived from various Chinese herbal medicines is a promising anticoccidiosis agent, but its therapeutic effects have not been comprehensively evaluated. This study aimed to assess the therapeutic efficacy of ChangQing Compound against Eimeria tenella-induced coccidiosis in chickens on the basis of physiological indicators, cecum lesions, and changes in microbial diversity. The comparison with the positive control group revealed the average weight gain (AWG) and anticoccidial index (ACI) of the chicks were significantly higher, in contrast, the feed conversion ratio (FCR), cecal lesion score (CLS), and oocyst count per gram of cecal content (OPG) were significantly lower (P<0.05). Notably, AWG (138.87 g), OPG (0.57 × 106), ACI (177.92), and FCR (2.51) reflected the significant therapeutic effect of the 2.5 g/L ChangQing compound treatment (CQM). Histological sections showed that the cecal villus damage and intestinal wall swelling were minimal in the CQM, consistent with the CLS (0.73). Additionally, the 2.5 g/L ChangQing compound treatment effectively prevented the decrease of red blood cells, platelets, and hemoglobin, while promoting the release of anti-inflammatory factors interleukin-10 and interleukin-4, and inhibiting the pro-inflammatory factors interferon-γ and interleukin-17. The microbial community structure in the CQM was most similar to that of the negative control group. In summary, ChangQing compound had multiple positive effects (e.g., promoting weight gain, alleviating anemia, suppressing coccidial proliferation, reducing intestinal damage, modulating immunity, and maintaining intestinal microbiota homeostasis). The study results may be relevant to developing a novel strategy for the clinical management of coccidiosis.

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 high­performance 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-1ß), 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-1ß, 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.

Anti-Eimeria tenella activity of Ethanamizuril in vitro and in vivo.

The prevalence of chicken coccidiosis in the poultry industry is a significant concern, further exacerbated by the emergence of drug-resistant coccidia resulting from the indiscriminate use of medications. Ethanamizuril, a novel triazine anti-coccidial compound, has been used to combat drug resistance. Currently, it is known that Ethanamizuril acts on the second-generation merozoites and early gametogenesis stages of Eimeria. Limited information exists regarding its impact on the early merozoites and exogenous stage of Eimeria. In the present study, the anti-coccidial properties of Ethanamizuril were evaluated both in vitro and in vivo. The in vitro experiments demonstrated that Ethanamizuril effectively inhibits the sporulation of E. tenella oocysts in a dose-dependent manner and significantly reduces the sporozoite excystation rate. Furthermore, in vivo tests revealed that treatment with 10 mg/L Ethanamizuril in drinking water significantly decreased the copy number of first-generation and secondary-generation merozoites in the chicken cecum, indicating that it can inhibit the development of whole schizonts development. Moreover, treatment with Ethanamizuril demonstrated excellent protective efficacy with an anti-coccidial index (ACI) of 180.2, which was manifested through higher body weight gains, lighter cecal lesion, lower fecal oocyst shedding score and reduced liver index. Collectively, this study suggests that Ethanamizuril effectively treats E. tenella infection by inhibiting both endogenous and exogenous stages development.

Effects of prebiotic (lactoferrin) and diclazuril on broiler chickens experimentally infected with Eimeria tenella.

Introduction: Avian coccidiosis presents a significant challenge to the poultry industry in Egypt, highlighting the urgent need for validating new drug targets offering promising prospects for the development of advanced anticoccidials. Although numerous reports highlight the activity of lactoferrin (LF) against various microorganisms, its potential against Eimeria has not been explored. The present study evaluated the potential anticoccidial effect of LF and diclazuril in broiler chickens experimentally infected with Eimeria tenella. Methods: A total of 100 one-day-old broiler chicks were divided into five equal groups (20 each) as follows: Group 1 (G1) served as the normal healthy control group, Group 2 (G2) consisted of chickens infected with 1 × 105 sporulated E. tenella oocysts at 14 days of age, Group 3 (G3) comprised infected chickens treated with diclazuril (0.5 mL/L in drinking water) for 3 days successively, Group 4 (G4) included infected chickens treated with LF (at a dose of 250 mg/kg of diet) from one day of age until the end of the study, and Group 5 (G5) comprised infected chickens treated with both LF and diclazuril. Results: The positive control group (G2) experienced significant reductions in body weight (BW), BW gain, serum glucose, lipase, amylase, total antioxidant capacity, several hematological indices, and total proteins, along with alterations in various antioxidant enzymes. Conversely, serum levels of aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatases (ALP), urea, creatinine, nitric oxide, mean corpuscular volume (MCV), White blood cells (WBCs), heterophils, alpha 2, beta 1, and liver contents of malondialdehyde were elevated in this group. Moreover, higher oocyst counts and lesion scores, along with histopathological alterations, were observed in G2. Remarkably, treatment with diclazuril and/or LF demonstrated potent antioxidant and anticoccidial effects, resulting in reduced shedding of oocysts, lesion scores, and lymphocytic infiltrates in the cecum. Additionally, these treatments improved the antioxidant and immune systems in chickens and restored all histopathological changes reported in the infected non-treated group (G2). Conclusion: This study offers novel perspectives on the potential anticoccidial effects of the combination of LF and diclazuril in broiler chickens infected with E. tenella, highlighting the potential synergistic actions of LF in treating poultry coccidiosis.

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.

Oral vaccination with a recombinant Lactobacillus plantarum expressing the Eimeria tenella rhoptry neck 2 protein elicits protective immunity in broiler chickens infected with Eimeria tenella.

BACKGROUND: Chicken coccidiosis is a protozoan disease that leads to considerable economic losses in the poultry industry. Live oocyst vaccination is currently the most effective measure for the prevention of coccidiosis. However, it provides limited protection with several drawbacks, such as poor immunological protection and potential reversion to virulence. Therefore, the development of effective and safe vaccines against chicken coccidiosis is still urgently needed. METHODS: In this study, a novel oral vaccine against Eimeria tenella was developed by constructing a recombinant Lactobacillus plantarum (NC8) strain expressing the E. tenella RON2 protein. We administered recombinant L. plantarum orally at 3, 4 and 5 days of age and again at 17, 18 and 19 days of age. Meanwhile, each chick in the commercial vaccine group was immunized with 3 × 102 live oocysts of coccidia. A total of 5 × 104 sporulated oocysts of E. tenella were inoculated in each chicken at 30 days. Then, the immunoprotection effect was evaluated after E. tenella infection. RESULTS: The results showed that the proportion of CD4+ and CD8+ T cells, the proliferative ability of spleen lymphocytes, inflammatory cytokine levels and specific antibody titers of chicks immunized with recombinant L. plantarum were significantly increased (P < 0.05). The relative body weight gains were increased and the number of oocysts per gram (OPG) was decreased after E. tenella challenge. Moreover, the lesion scores and histopathological cecum sections showed that recombinant L. plantarum can significantly relieve pathological damage in the cecum. The ACI was 170.89 in the recombinant L. plantarum group, which was higher than the 150.14 in the commercial vaccine group. CONCLUSIONS: These above results indicate that L. plantarum expressing RON2 improved humoral and cellular immunity and enhanced immunoprotection against E. tenella. The protective efficacy was superior to that of vaccination with the commercial live oocyst vaccine. This study suggests that recombinant L. plantarum expressing the RON2 protein provides a promising strategy for vaccine development against coccidiosis.

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.

Susceptibility and cecal microbiota alteration to Eimeria-infection in Yellow-feathered broilers, Arbor Acres broilers and Lohmann pink layers.

Coccidiosis, which is caused by Eimeria species, results in huge economic losses to the poultry industry. Arbor Acres (AA) broilers and yellow-feathered broilers are the dominant broilers in northern and southern China, respectively. However, their susceptibility to coccidiosis has not been fully compared. In this study, the susceptibility of yellow-feathered broilers, AA broilers and Lohmann pink layers to E. tenella was evaluated based on mortality rate, relative body weight gain rate, intestinal lesion score, oocyst output, anticoccidial index (ACI), and cecum weight and length. The yellow-feathered broilers were shown to produce significantly fewer oocysts with higher intestinal lesion score compared to AA broilers, which had the highest growth rates and ACI scores. Subsequently, changes in the cecal microbiota of the 3 chicken lines before and after high-dose infection (1 × 104 oocysts) with E. tenella were determined by 16S rRNA sequencing. The results showed that composition of the microbiota changed dramatically after infection. The abundance of Firmicutes and Bacteroidetes in the infected chickens decreased, and Proteobacteria increased significantly among the different chicken lines. At the genus level, Escherichia increased significantly in all 3 groups of infected chickens, but Lactobacillus decreased to 0% in the infected yellow-feathered broilers. The results of the study indicate that the susceptibility to E. tenella varies among the 3 chicken lines, and that changes in intestinal microbiota by E. tenella-infection among the different chicken lines had a similar trend, but to different degrees. This study provides basic knowledge of the susceptibility in the 3 chicken lines, which can be helpful for the control and prevention of coccidiosis.

Molecular characterization and functional analysis of Eimeria tenella ankyrin repeat-containing protein.

Chicken coccidiosis causes disastrous losses to the poultry industry all over the world. Eimeria tenella is the most prevalent of these disease-causing species. Our former RNA-seq indicated that E. tenella ankyrin repeat-containing protein (EtANK) was expressed differently between drug-sensitive (DS) and drug-resistant strains. In this study, we cloned EtANK and analyzed its translational and transcriptional levels using quantitative real-time PCR (qPCR) and western blotting. The data showed that EtANK was significantly upregulated in diclazuril-resistant (DZR) strain and maduramicin-resistant (MRR) strain compared with the drug-sensitive (DS) strain. In addition, the transcription levels in the DZR strains isolated from the field were higher than in the DS strain. The translation levels of EtANK were higher in unsporulated oocysts (UO) than in sporozoites (SZ), sporulated oocysts (SO), or second-generation merozoites (SM), and the protein levels in SM were significantly higher than in UO, SO, and SZ. The results of the indirect immunofluorescence localization showed that the protein was distributed mainly at the anterior region of SZ and on the surface and in the cytoplasm of SM. The fluorescence intensity increased further with its development in vitro. An anti-rEtANK polyclonal antibody inhibited the invasive ability of E. tenella in DF-1 cells. These results showed that EtANK may be related to host cell invasion, required for the parasite's growth in the host, and may be involved in the development of E. tenella resistance to some drugs.

Gene cloning, expression, and enzyme kinetics analysis of Eimeria tenella 2- methylcitrate synthase.

In prokaryotes and lower eukaryotes, 2-methylcitrate cycle (2-MCC) is the main pathway for propionate decomposition and transformation, but little is known about the 2-MCC pathway of Eimeria tenella. The analysis of genomic data found that the coding gene of 2- methylcitrate synthase (EC 2.3.3.5, PrpC) exists in E. tenella, which is a key enzyme of 2-MCC pathway. Through the search analysis of the database (ToxoDB), it was found that ETH_ 00026655 contains the complete putative sequence of EtprpC. In this study, we amplified the ORF sequence of EtprpC based on putative sequence. Then, prokaryotic expression, enzyme activity and kinetic analysis was performed. The results showed that the EtprpC ORF sequence was 1272 bp, encoding a 46.3 kDa protein comprising 424 amino acids. Enzyme activity assays demonstrate linearity between the initial reaction rate (OD/min) and EtPrpC concentration (ranging from 1.5 to 9 µg/reaction), with optimal enzyme activity observed at 41°C and pH 8.0. The results of enzymatic kinetic analysis showed that the Km of EtPrpC for propionyl-CoA, oxaloacetic acid, and acetyl-CoA was 5.239 ± 0.17 mM, 1.102 ± 0.08 µM, and 5.999 ± 1.24 µM, respectively. The Vmax was 191.11 ± 19.1 nmol/min/mg, 225.48 ± 14.4 nmol/min/mg, and 370.02 ± 25.8 nmol/min/mg when EtPrpC concentration at 4, 6, and 8 µg, respectively. Although the ability of EtPrpC to catalyze acetyl-CoA is only 0.11% of its ability to catalyze propionyl-CoA, it indicates that the 2-MCC pathway in E. tenella is similar to that in bacteria and may have a bypass function in the TCA cycle. This study can provide the theoretical foundation for the new drug targets and the development of new anticoccidial drugs.

Effects of dietary water-soluble extract of rosemary supplementation on growth performance and intestinal health of broilers infected with Eimeria tenella.

This study was conducted to explore the effect of dietary supplementation of water-soluble extract of rosemary (WER) on growth performance and intestinal health of broilers infected with Eimeria tenella (E. tenella), and evaluate the anticoccidial activity of WER. 360 1-d-old Chinese indigenous male yellow-feathered broiler chickens were randomly allocated to six groups: blank control (BC) group and infected control (IC) group received a basal diet; positive control (PC) group, received a basal diet supplemented with 200 mg/kg diclazuril; WER100, WER200, and WER300 groups received a basal diet containing 100, 200, and 300 mg/kg WER, respectively. On day 21, all birds in the infected groups (IC, PC, WER100, WER200, and WER300) were orally gavaged with 1 mL phosphate-buffered saline (PBS) of 8 × 104 sporulated oocysts of E. tenella, and birds in the BC group were administrated an aliquot of PBS dilution. The results showed that dietary supplementation of 200 mg/kg WER increased the average daily gain of broilers compared to the IC group from days 22 to 29 (P < 0.001). The anticoccidial index values of 100, 200, and 300 mg/kg WER were 137.49, 157.41, and 144.22, respectively, which indicated that WER exhibited moderate anticoccidial activity. Compared to the IC group, the groups supplemented with WER (100, 200, and 300 mg/kg) significantly lowered fecal oocyst output (P < 0.001) and cecal coccidia oocysts, alleviated intestinal damage and maintained the integrity of intestinal epithelium. Dietary supplementation with WER significantly improved antioxidant capacity, elevated the levels of secretory immunoglobulin A, and diminished inflammation within the cecum, particularly at a dosage of 200 mg/kg. The results of this study indicated that dietary supplementation with 200 mg/kg WER could improve broiler growth performance and alleviate intestinal damage caused by coccidiosis.

Avian coccidiosis, a prevalent parasitic disease caused by Eimeria protozoa, leads to significant economic losses in the global poultry industry. Currently, the control of coccidiosis in chickens primarily relies on chemical and ionophore anticoccidials. However, the long-term use of these compounds has resulted in the development of drug-resistant strains, presenting a critical challenge. Additionally, the toxic and side effects of ionophore anticoccidials have become increasingly apparent. Thus, there is an urgent need to find economical and environmentally friendly measures to control coccidiosis in chickens. In this study, we established a model of Eimeria tenella infection in broilers to explore whether the water-soluble extract of rosemary (WER) could serve as an alternative method for controlling avian coccidiosis. Our results showed that dietary supplementation with WER (100, 200, and 300 mg/kg) had a beneficial anticoccidial effect, alleviating intestinal damage caused by coccidiosis by enhancing the intestinal antioxidant defense and activating the immune function of the infected broilers. Specifically, dietary supplementation with 200 mg/kg WER emerged as a promising strategy for controlling avian coccidiosis in the poultry industry.

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-1ß, 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.

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)-1ß (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.

Correlation of caecal microbiome endotoxins genes and intestinal immune cells in Eimeria tenella infection based on bioinformatics.

Introduction: The infection with Eimeria tenella (ET) can elicit expression of various intestinal immune cells, incite inflammation, disrupt intestinal homeostasis, and facilitate co-infection with diverse bacteria. However, the reciprocal interaction between intestinal immune cells and intestinal flora in the progression of ET-infection remains unclear. Objective: The aim of this study was to investigate the correlation between cecal microbial endotoxin (CME)-related genes and intestinal immunity in ET-infection, with subsequent identification of hub potential biomarker and immunotherapy target. Methods: Differential expression genes (DEGs) within ET-infection and hub genes related to CME were identified through GSE39602 dataset based on bioinformatic methods and Protein-protein interaction (PPI) network analysis. Moreover, immune infiltration was analyzed by CIBERSORT method. Subsequently, comprehensive functional enrichment analyses employing Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis along with Gene Ontology (GO), gene set enrichment analysis (GSEA), and gene set variation analysis (GSVA) were performed. Results: A total of 1089 DEGs and 25 hub genes were identified and CXCR4 was ultimately identified as a essential CME related potential biomarker and immunotherapy target in the ET-infection. Furthermore, activated natural killer cells, M0 macrophages, M2 macrophages, and T regulatory cells were identified as expressed intestinal immune cells. The functional enrichment analysis revealed that both DEGs and hub genes were significantly enriched in immune-related signaling pathways. Conclusion: CXCR4 was identified as a pivotal CME-related potential biomarker and immunotherapy target for expression of intestinal immune cells during ET-infection. These findings have significant implications in elucidating the intricate interplay among ET-infection, CME, and intestinal immunity.

Cloning, expression, and functional identification of aquaporin genes from Eimeria tenella.

Avian coccidiosis, caused by Eimeria spp., is one of the major parasitic diseases in chicken. Aquaporins (AQP) are essential mediators of water regulation and nutritional intake in parasites, and it may be a suitable molecule for chemotherapeutic target and vaccine candidate. We identified two aquaporin genes in Eimeria tenella (EtAQP1 and EtAQP2) with their full sequence, and the expression profiles were analyzed across different stages of E. tenella life cycle. The expression of EtAQP1 and EtAQP2 in Xenopus oocytes renders them highly permeable for both water and glycerol. Sugar alcohols up to five carbons and urea pass the pore. The immunohistochemical analysis confirms the restriction of antiserum staining to the surface of transfected Xenopus oocytes. Like other AQP family, EtAQPs are transmembrane proteins that are likely important molecules that facilitate solute uptake for parasite intracellular growth and therapeutic targets.