The impact of different levels of functional oil supplementation in combination with salinomycin on growth performance and intestinal microbiota of broilers undergoing Eimeria challenge: An analysis of dynamics.

The effect of salinomycin sodium alone and in combination with functional oils on performance and microbiota of broiler infected Eimeria were evaluated. 512 broilers were randomly assigned to 4 treatments (8 replicates, 16 birds/pen): a Control group (any additives); Ionophore group: salinomycin supplementation at 66 ppm (SS66); Ionophore +0.075% Functional oil (FO) group (SS66 + FO supplementation at 750 ppm); and Ionophore +0.10% FO group (SS66 + FO supplementation at 1000 ppm). At 14 days of age, birds were gavaged with 1 mL of a saline solution containing sporulated oocysts of E. tenella, E. acervulina and E. maxima. Performance indices were measured weekly. At 28 days, intestinal content was collected for microbiota analysis. Broilers of Control group presented the worst performance indices. Broilers of Ionophore + FO (0.075% and 0.10%) groups exhibited a higher BW at 28 days of age. The supplementation of Ionophore +0.075% FO resulted in a higher relative proportion of Firmicutes and a lower proportion of Actinobacteria in the ileum-jejunum. Lactobacillaceae was the dominant family in the jejunal, and ileal microbiotas of broilers fed diets supplemented with Ionophore, Ionophore +0.075% FO and Ionophore +0.10% FO. The supplementation of ionophore yielded higher numbers of Lactobacillaceae, Enterobactereaceae and Cloritridiaceae in the cecal. Ionophore associated with FO controlled the Lactobacillaceae, Enterobactereaceae and Cloritridiaceae families present in the cecum. Therefore, the combination of salinomycin with functional oil showed synergistic effect on performance and modulation of intestinal microbiota of broilers challenged with Eimeria.

Different methionine to cysteine supplementation ratios altered bone quality of broilers with or without Eimeria challenge assessed by dual energy X-ray absorptiometry and microtomography.

Despite the acknowledged significance of nutrition in bone development, effects of methionine (Met) and cysteine (Cys) on bone quality remain under-researched, particularly during Eimeria challenge. We investigated the effects of different supplemental Met to Cys ratios (MCR) on bone quality of broilers under Eimeria challenge. A total of 720 fourteen-day old Cobb500 broilers were allocated into a 5 × 2 factorial arrangement. Five diets with Met and Cys supplemented at MCR of 100:0, 75:25, 50:50, 25:75, and 0:100 were fed to the birds with or without Eimeria challenge. Body composition was measured by dual energy x-ray absorptiometry, and the femur bone characteristics were assessed by microtomography. Data were analyzed by two-way ANOVA and orthogonal polynomial contrast. The results reaffirmed the detrimental effects of Eimeria challenge on bone quality. On 9 d post inoculation (DPI), significant interaction effects were found for whole body bone mineral content (BMC), lean tissue weight, and body weight (P < 0.05); in the nonchallenged group (NCG), these parameters linearly decreased as MCR decreased (P < 0.05). In the challenged group (CG), body weight and lean tissue weight were unaffected by MCR, and BMC linearly increased as MCR decreased (P < 0.05). For the cortical bone of femoral metaphysis on 6 DPI, bone mineral density (BMD) linearly increased as MCR decreased (P < 0.05). Bone volume to tissue volume ratio (BV/TV) in the CG linearly increased as MCR decreased (P < 0.05). On 9 DPI, BMC and TV linearly increased as MCR decreased (P < 0.05) in the NCG. BMD and BV/TV changed quadratically as MCR decreased (P < 0.05). For the trabecular bone of femoral metaphysis on 9 DPI, BV/TV, and trabecular number linearly increased as MCR decreased (P < 0.05) in the NCG. For the femoral diaphysis, BV, TV, BMC on 6 DPI, and BMD on 9 DPI linearly increased as MCR decreased (P < 0.05). In conclusion, this study showed that both Eimeria challenge and varying supplemental MCR could influence bone quality of broilers.

Identification and characterization of the receptors of a microneme adhesive repeat domain of Eimeria maxima microneme protein 3 in chicken intestine epithelial cells.

Eimeria maxima microneme protein 3 (EmMIC3) is pivotal in the initial recognition and attachment of E. maxima sporozoites to host cells. EmMIC3 comprises 5 tandem Type I microneme adhesive repeat (MAR) domains, among which MAR2 of EmMIC3 (EmMAR2) has been identified as the primary determinant of EmMIC3-mediated tissue tropism. Nonetheless, the mechanisms through which EmMAR2 guides the parasite to its invasion site through interactions with host receptors remained largely uncharted. In this study, we employed yeast two-hybrid (YTH) screening assays and shotgun LC-MS/MS analysis to identify EmMAR2 receptors in chicken intestine epithelial cells. ATPase H+ transporting V1 subunit G1 (ATP6V1G1), receptor accessory protein 5 (REEP5), transmembrane p24 trafficking protein (TMED2), and delta 4-desaturase sphingolipid 1 (DEGS1) were characterized as the 4 receptors of EmMAR2 by both assays. By blocking the interaction of EmMAR2 with each receptor using specific antibodies, we observed varying levels of inhibition on the invasion of E. maxima sporozoites, and the combined usage of all 4 antibodies resulted in the most pronounced inhibitory effect. Additionally, the spatio-temporal expression profiles of ATP6V1G1, REEP5, TMED2, and DEGS1 were assessed. The tissue-specific expression patterns of EmMAR2 receptors throughout E. maxima infection suggested that ATP6V1G1 and DEGS1 might play a role in early-stage invasion, whereas TMED2 could be involved in middle and late-stage invasion and REEP5 and DEGS1 may participate primarily in late-stage invasion. Consequently, E. maxima may employ a multitude of ligand-receptor interactions to drive invasion during different stages of infection. This study marks the first report of EmMAR2 receptors at the interface between E. maxima and the host, providing insights into the invasion mechanisms of E. maxima and the pathogenesis of coccidiosis.

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.

Defining optimal dietary starch, oil, and amino acid inclusion levels for broilers experiencing a coccidiosis challenge.

Alternative methods to alleviate coccidiosis in broilers are of interest to producers, including dietary strategies to minimize disruptions in growth rate and efficiency when faced with health challenges. Our objective was to determine optimal combinations of dietary starch, amino acids (AA), and oil to benefit productivity of broilers experiencing Eimeria-induced immune activation. Two trials were conducted using 1,536 male Ross 308 broiler chicks in floor pens randomly assigned to 1 of 17 experimental treatments. All birds received common starter (d 0-10) and finisher (d 24-35) diets, and only differed based on their assigned experimental grower diet (d 10-24). Trial 1 experimental grower diets ranged from 2,700 to 3,300 kcal/kg AME. Trial 2 included 10 experimental grower diets following a simplex lattice design consisting of 3 basal lots formulated to have the highest starch (45.4%), oil (10.2%), or AA density (120, 1.33% digestible Lys) and mixed in 4 equally spaced levels for each component (0, 0.33, 0.67, 1). These mixtures enabled varying densities of AA (80-120% of recommendation), starch:oil (4:1-20:1), and AME (2,940-3,450 kcal/kg). Bird and feeder weights were collected on d 0, 10, 24, and 35, and birds were exposed to an Eimeria challenge on d 11 or 12. In trial 2, excreta samples were collected for AME determination and carcasses were processed on d 36. Data were analyzed using ANOVA, t test, or regression. In Trial 1, BW gain and feed conversion were improved (P < 0.05) by increasing dietary AME. In Trial 2, birds receiving diets containing AA at 93 to 107% of recommendations and higher oil exhibited improved (P < 0.05) performance, but increased starch at the expense of oil reduced performance (P < 0.05). Relative breast and fat pad weights were not influenced by diet in Trial 2. We determined that broilers mildly challenged with Eimeria would exhibit highest BW gain when receiving diets containing 35.8% starch, 8.9% oil, and 101.3% of AA recommendations, which can be utilized by producers to maintain productivity under health-challenged conditions.

Impacts of varying methionine to cysteine supplementation ratios on growth performance, oxidative status, intestinal health, and gene expression of immune response and methionine metabolism in broilers under Eimeria spp. challenge.

A study was conducted to investigate effects of different methionine (Met) to cysteine (Cys) supplementation ratios (MCR) on growth performance, oxidative status, intestinal health, immune responses, and methionine metabolism in broilers under Eimeria challenge. A total of 720 male Cobb500 broilers (14-day-old) were allocated in a 2 × 5 factorial arrangement (5 diets, with or without challenge) with 6 replicates per treatment. The total sulfur amino acid concentrations were consistent across treatments meeting the breeder's recommendation, only MCR varied. The diets were labeled as MET100; MET75; MET50; MET25; and MET0, representing MCR of 100:0; 75:25; 50:50; 25:75; and 0:100, respectively. Data were analyzed by 2-way ANOVA and orthogonal polynomial contrast. Growth performance declined linearly or quadratically as MCR decreased (P < 0.01). On 6-day postinoculation (DPI), interaction effects (P < 0.01) were found; BW and body weight gain were lower in MET0 compared to the other treatments in the nonchallenged groups, whereas not in the challenged groups. On 6 and 9 DPI, serum total antioxidant capacity linearly decreased as MCR decreased (P < 0.05). Hepatic activities of glutathione peroxidase on 6 DPI and superoxide dismutase on 9 DPI changed quadratically as MCR decreased (P < 0.05). The digestibility of Met linearly decreased whereas the digestibility of Cys linearly increased as MCR decreased. The ileal crypt depth linearly decreased as MCR decreased (P < 0.01) on 6 DPI. The expression of transforming growth factor beta on 6 and 9 DPI, tumor necrotic factor alpha and interleukin 10 on 9 DPI changed quadratically as MCR decreased (P < 0.05). Eimeria challenge increased expression of Met adenosyltransferase and cystathionine gamma-lyase, whereas decreasing the expression of other Met metabolism genes (P < 0.01) on 6 DPI. Expression of Met metabolism genes linearly increased as MCR decreased (P < 0.05). In conclusion, different Met to Cys supplementation ratios exerted linearly or quadratically effects on the growth performance, oxidative status, intestinal health, and metabolism of Met in broiler chickens under Eimeria infection.

IFN-γ inhibitory molecules derived from Eimeria maxima inhibit IL-12 secretion by modulating MAPK pathways in chicken macrophages.

IFN-γ plays a crucial role in resisting intracellular parasitic protozoa, such as Eimeria species. In our previous study, we identified 4 molecules derived from Eimeria maxima (E. maxima) that significantly inhibited IFN-γ production. However, the mechanism underlying this inhibitory effect remains unknown. In this study, we first investigated the effects of these 4 IFN-γ inhibitory molecules on the expression levels of chicken Toll-like receptors (chTLRs), IL-12, IL-10, TGF-ß, and TNF-α in chicken macrophage HD11 and bone marrow-derived dendritic cells (BMDCs). The results demonstrated that these 4 inhibitory molecules significantly downregulated the mRNA levels of chTLR-2, chTLR-4, chTLR-21, and both mRNA and protein levels of IL-12. Subsequently, to clarify the effects of these 4 inhibitory molecules on the IL-12 secretion-related signaling pathways in chicken macrophages, qRT-PCR and Western blot were used to detect the changes of key molecules involved in the signaling pathways of IL-12 secretion (NF-κB, ERK1/2, p38, JNK, STAT3) following coincubation with these inhibitory molecules. Finally, RNAi was employed to verify the function of key molecules in the signaling pathway. The results revealed a significant upregulation in the expression of ERK1/2 phosphorylated protein induced by the 4 inhibitory molecules. Knockdown of the ERK1/2 gene significantly reduced the inhibitory effect of the 4 E. maxima inhibitory molecules on IL-12. These findings indicate that the 4 inhibitory molecules can inhibit the secretion of IL-12 by upregulating the expression of ERK1/2 phosphorylated protein, which is a key molecule in the ERK-MAPK pathway. Our study may contribute to elucidating the mechanisms underlying immune evasion during E. maxima infections, thereby providing new insights for the control of chicken coccidiosis.

Proteomic Analysis of Fractionated Eimeria tenella Sporulated Oocysts Reveals Involvement in Oocyst Wall Formation.

Eimeria tenella is the most pathogenic intracellular protozoan parasite of the Eimeria species. Eimeria oocyst wall biogenesis appears to play a central role in oocyst transmission. Proteome profiling offers insights into the mechanisms governing the molecular basis of oocyst wall formation and identifies targets for blocking parasite transmission. Tandem mass tags (TMT)-labeled quantitative proteomics was used to analyze the oocyst wall and sporocysts of E. tenella. A combined total of 2865 E. tenella proteins were identified in the oocyst wall and sporocyst fractions; among these, 401 DEPs were identified, of which 211 were upregulated and 190 were downregulated. The 211 up-regulated DEPs were involved in various biological processes, including DNA replication, fatty acid metabolism and biosynthesis, glutathione metabolism, and propanoate metabolism. Among these proteins, several are of interest for their likely role in oocyst wall formation, including two tyrosine-rich gametocyte proteins (EtGAM56, EtSWP1) and two cysteine-rich proteins (EtOWP2, EtOWP6). Concurrently, 96 uncharacterized proteins may also participate in oocyst wall formation. The present study significantly expands our knowledge of the proteome of the oocyst wall of E. tenella, thereby providing a theoretical basis for further understanding of the biosynthesis and resilience of the E. tenella oocyst wall.

Supplementing broiler diets with bacterial selenium nanoparticles enhancing performance, carcass traits, blood indices, antioxidant status, and caecal microbiota of Eimeria tenella-infected broiler chickens.

Nanomedicine is a critical therapeutic approach for treating most poultry illnesses, particularly parasitic infections. Coccidiosis is a severe protozoan infection affecting poultry; the emergence of drug-resistant Eimeria strains demands the development of new, safe therapies. Consequently, the objective of this work was to investigate the efficacy of the biosynthesized selenium nanoparticles (SeNPs) by Paenibacillus polymyxa (P. polymyxa) against Eimeria tenella (E. tenella) experimental infection in broiler chickens. The prepared SeNPs absorbed the UV at 270 nm were spherical with a size of 26 nm, and had a surface negative charge of -25 mV. One hundred and fifty, 1-day-old male broiler chicks were randomly allocated into 5 groups (30 birds/group with triplicates each) as follows: T1: negative control (noninfected and nontreated with SeNPs); T2: delivered SeNPs (500 µg/kg diet) for 35 successive days, T3: E. tenella-infected (positive control birds), T4: E. tenella-infected and treated with SeNPs (500 µg/kg diet) and T5: E. tenella-infected chicks and treated with anticoccidial agent (sulfadimidine, 16% solution 8 mL/L of drinking water) for 5 successive days. At 14 d of age, each bird in infected groups was orally treated with 3 × 103 sporulated oocyst of E. tenella. SeNPs considerably decreased the number of oocysts in broiler feces compared to positive control and anticoccidial drug, followed by a substantial reduction of parasite phase count in the cecum (15, 10, and 8 for meronts, gamonts, and developing oocysts) when compared with positive control birds. The Eimeria experimental infection lowered the activity of antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), and reduced glutathione (GSH) while increasing the stress parameters nitric oxide (NO) and malonaldehyde (MDA). Moreover, the production of proinflammatory (TNF-α and IL-6) and apoptotic genes (BcL2 and Cas-3) were significantly elevated. Administrating SeNPs to chicks significantly decreased oxidative stress, inflammation, and apoptotic markers in the cecum tissue. Therefore, growth performance, carcass weights, antioxidant enzymes, and blood properties of infected chicks were enhanced. The findings compared the protecting role of Se-nanoparticles against cecum damages in E. tenella-infected broilers.

Dynamics of protozoal excretion in the faeces of calves during the first 28 days after arrival at the fattening farm indicate infection before regrouping and show poor temporal correlation with diarrhoea.

BACKGROUND: Calves in dairy cattle production in Switzerland are transported to a fattening farm at the age of 3-5 weeks, and frequently suffer from diarrhoea within the first 14 days after arrival. To characterise the role of intestinal protozoa in this, we investigated the excretion dynamics of Eimeria, Cryptosporidium and Giardia during the first 28 days after the arrival and regrouping of calves at fattening farms. METHODS: A total of 610 faecal samples from 122 calves (mean age 37.3 days; mean body weight 79.8 kg) were collected on seven different fattening farms during the first 28 days after the arrival and regrouping of the animals. The farms were visited between January and April (cold season; n = 4) and between June and August (warm season; n = 3). The samples were collected rectally on days 1, 4, 7, 14 and 28, assessed for consistency, and analysed using the McMaster method for quantitative determination of the number of Eimeria oocysts per gram of faeces (OPG), flotation for morphological differentiation of the unsporulated Eimeria oocysts, a concentration method for the semi-quantitative determination of Giardia cysts, and modified Ziehl-Neelsen staining for semi-quantitative determination of Cryptosporidium oocysts. RESULTS: Overall, 50.8% (62/122) of the animals had diarrhoea during the study period. However, the faecal excretion of protozoal pathogens was neither associated with diarrhoea nor with body weight gain of the animals. Altogether, 90.2% (110/122) of the calves were Eimeria positive. Eimeria zuernii was excreted by 51 (41.8%) and Eimeria bovis by 68 (55.7%) animals. In the warm season more animals tested positive for Eimeria and OPGs were higher than in the cold season. There was no correlation between the age of the calves and the OPG values. Overall, 64.8% (79/122) of the calves excreted Eimeria oocysts within the first 7 days, indicating that they had been infected with the parasite on the dairy farm of origin. Eighty-nine calves (73.0%) excreted Giardia cysts, with more positive animals in the cold (80.3%) compared with the warm season (64.3%). Only Giardia duodenalis assemblage E was identified. Cryptosporidium oocysts were microscopically detected in 14 animals (11.5%) on five farms. Cryptosporidium spp. were present in a total of 12 animals, i.e. Cryptosporidium parvum in nine, Cryptosporidium ryanae in two, and Cryptosporidium bovis in one animal. CONCLUSIONS: A better understanding of the temporal dynamics of protozoal infections in calves is helpful for the implementation of appropriate measures to protect the health of these animals at a critical phase in their lives. Our results indicate that factors other than those examined in the present study contributed to the onset of diarrhoea in the calves.

Intestinal health of broilers challenged with Eimeria spp. using functional oil blends in two physical forms with or without anticoccidials.

This study aimed to assess the impact of a commercial blend of functional oils, specifically cashew nutshell liquid and castor oil (FO), in two physical forms (solid: P; liquid: S), in comparison to a combination of virginiamycin and anticoccidials on the gut health of broilers challenged with coccidiosis. A total of 1760 1-day-old male chicks were randomly distributed in a study design with eight treatments. The treatments included: a control group (without additive), OFS_0.75_kg/t (FO spray), OFP_1.0_kg/t (FO powder), OFP_1.5_kg/t (FO liquid spray), Sal (anticoccidials), Sal_Vir (virginiamycin and anticoccidials), Sal_OFS_0.5_ kg/t (anticoccidials plus FO spray), and Sal_OFP_1.0_kg/t (anticoccidials plus FO powder). All birds were challenged with Eimeria spp. at 14 days. The physical form of FO did not affect performance and intestinal health parameters. At 42 days, broilers from the control and OFS_0.75 treatments were the lightest, while those from the Sal_Vir and Sal_OFP_1.0 treatments were the heaviest (P < 0.05). FO reduced the presence of Clostridium perfringens. The individual phytogenic additives did not prevent weight loss in birds challenged with Eimeria, but they mitigated the effects of the infection by modulating the intestinal microbiota. A synergistic effect was observed between the FO and anticoccidials, yielding satisfactory results in substituting virginiamycin.

Immune modulation of Th1/Th2/Treg/Th17/Th9/Th21 cells in rabbits infected with Eimeria stiedai.

Introduction: Despite long-term integrated control programs for Eimeria stiedai infection in China, hepatic coccidiosis in rabbits persists. Th1, Th2, Th17, Treg, Th9, and Th21 cells are involved in immune responses during pathogen infection. It is unclear whether Th cell subsets are also involved in E. stiedai infection. Their roles in the immunopathology of this infection remain unknown. Therefore, monitoring these T-cell subsets' immune responses during primary infection of E. stiedai at both transcriptional (mRNA) and protein (cytokines) levels is essential. Methods: In experimentally infected New Zealand white rabbits, mRNA expression levels of their transcript-TBX2 (Th1), GATA3 (Th2), RORC (Th17), Foxp3 (Treg), SPI1 (Th9), and BCL6 (Th21)-were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR), whereas Th1 (IFN-g and TNF-a), Th2 (IL4), Th17 (IL17A and IL6), Treg (IL10 and TGF-b1), Th9 (IL9), and Th21 (IL21) cytokines were measured using enzyme-linked immunosorbent assays (ELISAs). Results: We found that levels of TBX2, GATA3, RORC, SPI1, and BCL6 in the livers of infected rabbits were elevated on days 5 and 15 post-infection (PI). The concentrations of their distinctive cytokines IFN-g and TNF-a for Th1, IL4 for Th2, IL17A for Th17, IL9 for Th9, IL21 for Th21, and IL10 for Treg IL10 were also significantly increased on days 5 and 15 PI, respectively (p < 0.05). On day 23 PI, GATA3 with its cytokine IL4, RORC with IL17A, Foxp3 with IL10 and TGF-b1, and SPI1 with IL9 were significantly decreased, but TBX2 with IFN-g and IL6 remained elevated. Discussion: Our findings are the first evidence of Th1/Th2/Treg/Th17/Th9/Th21 changes in E. stiedai-infected rabbits and provide insights into immune regulation mechanisms and possible vaccine development.

Responses of broilers challenged by Eimeria maxima fed with different levels of dietary balanced protein.

This study aimed to evaluate the effects of different dietary balanced protein (BP) levels on the gut health, amino acid apparent ileal digestibility (AID), footpad dermatitis lesions, and litter quality in broiler chicks infected with Eimeria maxima. A total of 2400 male 14-day-old Cobb500 broilers were randomly allotted into 10 treatments with six replications containing 40 birds each in a factorial design of 5 × 2. The treatments consisted of five levels of BP (6.66%, 13.32%, 19.98%, 26.64%, and 33.3%), and broilers unchallenged (NCH) or challenged (CH). Broilers in the CH group received 1 mL of Eimeria maxima inoculum (7 × 103 sporulated oocysts/mL). Oocyst count in excreta, visual intestinal modifications score, morphology, and morphometrics of the ileum were used to determine gut health status. Additionally, amino acids and CP AID, litter quality, and footpad dermatitis were evaluated. An ANOVA and Kruskal-Wallis tests followed by post-hoc tests were performed. The oocyst count in the CH group increased with an increase in dieatary BP (P = 0.08). The incidence of intestinal modifications was higher in the CH group (P < 0.05) and increased with increasing dietary BP (P < 0.05). Morphometrics were impaired by the challenge (P < 0.05), and by the two highest BP levels (P < 0.05). Amino acids AID (methionine, methionine + cystine, arginine, and serine) were reduced by E. maxima challenge. An increase in dietary BP resulted in poor litter quality and high prevalence of of footpad dermatitis (P < 0.05). The E. maxima challenge and increased BP decreased gut health, litter quality, and cause a high incidence of footpad dermatitis.

Effects of dietary essential oil supplementation on growth performance, carcass yield, meat quality, and intestinal tight junctions of broilers with or without Eimeria challenge.

The effects of dietary supplementation of essential oil on growth performance, carcass yield, meat quality, serum antioxidant capacity, and intestinal tight junctions of broilers with or without Eimeria challenge were investigated. A total of 576 one-day-old male broilers were randomly separated into 8 treatments (6 replication floor-pens per treatment, 12 broilers per pen) in a 4 × 2 factorial design. The 4 diets consisted of 1) a corn and soybean meal basal diet, 2) an anticoccidial diet (60 g nicarbazin and 60 g narasin per ton of feed), 3) an oregano oil diet (500 ppm oregano oil), and 4) a clove oil diet (500 ppm clove oil). On d 10, half chicks were challenged with 1 × 104 sporulated oocysts of E. tenella, E. acervulina, and E. maxima per chick, whereas the others were inoculated with an equal amount of dilution (0.5 mL). The Eimeria challenge induced a higher fecal oocyst output on d 18, a lower duodenum Occludin expression level on d 28, a lower serum catalase level, and a higher cook loss and protein loss in thigh muscle on d 42. The anticoccidial diet lowered fecal Eimeria output and increased d 1 to 42 BW gain as compared to the control diet. The clove oil treatment enhanced duodenum ZO-1 expression level in nonchallenged birds, increased BW gain from d 1 to 14 and breast yield on d 42. The oregano oil treatment enhanced ZO-1 expression of challenged birds, reduced feed intake from 15 to 28 d, and helped broilers gain more tender meat. For those Eimeria-challenged broilers, both clove and oregano oil treatments recovered drip loss in breast muscle. Our results suggested that Eimeria challenge in broiler early age could interrupt later serum antioxidant capacity and damage meat quality. The dietary supplementation of clove or oregano essential oils could improve broiler growth performance and partially relieve the coccidial damage in gut integrity and meat quality.

Forward genetic analysis of monensin and diclazuril resistance in Eimeria tenella.

Worldwide distributed coccidiosis is caused by infection of both Eimeria species and Cystoisospora in the host intestine and causes huge economic losses to the livestock industry, especially the poultry industry. The control of such diseases relies mainly on chemoprophylaxis with anticoccidials, which has led to a very common drug resistance in this field. However, the genetic mechanisms underlying resistance to many anticoccidial drugs remain unknown. In this study, strains of E. tenella resistant to 250 mg/kg monensin were generated and characterized. Forward genetic approaches based on pooled genome sequencing, including experimental evolution and linkage group selection, were used to locate candidate targets responsible for resistance to monensin and diclazuril in E. tenella. A total of 16 nonsynonymous mutants in protein-coding genes were identified in monensin-resistant strains, and two genomic regions with strong selection signals were also detected in diclazuril-resistant strains. Our study reveals the genetic characterization of the experimental evolution and linkage group selection in Eimeria species, and also provides important information that contributes to the understanding of the molecular mechanism of drug resistance in coccidia.

Evaluation of a dacitic (rhyolitic) tuff breccia use on performance, inflammatory, and antioxidant responses in broilers mildly challenged with Eimeria spp.

The objective of the study was to investigate the effects of a dacitic tuff breccia (DTB) on Eimeria-infected broilers. A total of 600 one-day-old Cobb 500 male chickens were randomly assigned to 5 treatments with 10 replicates of 12 birds. Treatments were: an unchallenged control (UC), a challenged (CC) control (0% DTB), and 3 challenged groups with 0.125, 0.25, or 0.5% DTB. At d 14, birds in the CC and DTB groups were orally gavaged with mixed Eimeria spp., while the UC received water. Growth performance was evaluated during prechallenge, challenge, and postchallenge periods (0-14 d; 14-20 d; and 20-26 d, respectively). Gastrointestinal permeability was measured at 5 days postinfection (dpi). Intestinal histology and nutrient digestibility of dry matter (DM), crude protein (CP), and ileal digestible energy (IDE) were measured at 6 dpi. Liver activity of glutathione peroxidase (GSH-Px) was determined at 6 dpi, and concentrations of reduced (GSH) and oxidized glutathione (GSSG) were analyzed at 6 and 12 dpi. Data were analyzed using a linear mixed model analysis and Tukey's test (P ≤ 0.05). From 0 to 14 d, similar average daily gain (ADG) and average daily feed intake (ADFI, P > 0.05) were observed. Gain:feed ratio (GF) was higher in 0.125, 0.25, and 0.5% of DTB than the CC and UC (P < 0.001). From 14 to 20 d, the UC had the highest ADG, ADFI, and GF (P < 0.001). At 5 dpi, intestinal permeability was higher in the challenged groups than the UC. Additionally, the UC showed the highest apparent ileal digestibility of CP, whereas 0.125% DTB had higher CP digestibility than the CC and 0.5% DTB (P < 0.001). At 6 dpi, 0.125% DTB increased GSH-Px activity compared to the CC, 0.5% DTB, and UC (P < 0.001). At 12 dpi, 0.125% DTB showed increased GSH concentration compared to the CC, 0.25% DTB, and 0.5% DTB (P < 0.01). The mild coccidia infection negatively impacted growth performance, apparent ileal nutrient digestibility, intestinal histology, and gastrointestinal integrity in broilers. The use of 0.125% DTB exhibited potential in improving antioxidant responses, apparent ileal digestibility of CP, and growth performance.

In Vitro Transcriptional Response of Eimeria tenella to Toltrazuril Reveals That Oxidative Stress and Autophagy Contribute to Its Anticoccidial Effect.

Intestinal coccidiosis is a common parasitic disease in livestock, caused by the infection of Eimeria and Cystoisospora parasites, which results in great economic losses to animal husbandry. Triazine compounds, such as toltrazuril and diclazuril, are widely used in the treatment and chemoprophylaxis of coccidiosis. Unfortunately, widespread drug resistance has compromised their effectiveness. Most studies have focused on prophylaxis and therapeutics with toltrazuril in flocks, while a comprehensive understanding of how toltrazuril treatment alters the transcriptome of E. tenella remains unknown. In this study, merozoites of E. tenella were treated in vitro with 0.5 µg/mL toltrazuril for 0, 1, 2 and 4 h, respectively. The gene transcription profiles were then compared by high-throughput sequencing. Our results showed that protein hydrolysis genes were significantly upregulated after drug treatment, while cell cycle-related genes were significantly downregulated, suggesting that toltrazuril may affect parasite division. The expression of redox-related genes was upregulated and elevated levels of ROS and autophagosomes were detected in the parasite after toltrazuril treatment, suggesting that toltrazuril may cause oxidative stress to parasite cells and lead to its autophagy. Our results provide basic knowledge of the response of Eimeria genes to toltrazuril and further analysis of the identified transcriptional changes can provide useful information for a better understanding of the mechanism of action of toltrazuril against Eimeria.

In vivo anticoccidial, antioxidant, and anti-inflammatory activities of avocado fruit, Persea americana (Lauraceae), against Eimeria papillata infection.

Apicomplexan parasites, especially Eimeria sp., are the main intestinal murine pathogens, that lead to severe injuries to farm and domestic animals. Many anticoccidial drugs are available for coccidiosis, which, leads to the development of drug-resistant parasites. Recently, natural products are considered as an alternative agent to control coccidiosis. This study was designed to evaluate the anticoccidial activity of the Persea americana fruit extract (PAFE) in male C57BL/6 mice. A total of 35 male mice were divided into seven equal groups (1, 2, 3, 4, 5, 6, and 7). At day 0, all groups except the first group which served as uninfected-untreated control were infected orally with 1 × 103E. papillata sporulated oocysts. Group 2 served as uninfected-treated control. Group 3 was considered an infected-untreated group. After 60 min of infection, groups 4, 5, and 6 were treated with oral doses of PAFE aqueous methanolic extract (100, 300, and 500 mg/kg of body weight, respectively). Group 7 was treated with amprolium (a reference drug for coccidiosis). PAFE with 500 mg/kg, was the most effective dose, inducing a significant reduction in the output of oocysts in mice feces (by about 85.41%), accompanied by a significant decrease in the number of the developmental parasite stages and a significant elevation of the goblet cells in the jejunal tissues. Upon treatment, a significant change in the oxidative status due to E. papillata infection was observed, where the levels of glutathione (GSH) increased, while, levels of malondialdehyde (MDA) and nitric oxide (NO) were decreased. In addition, the infection significantly upregulated the inflammatory cytokines of interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-α), and interferon-γ (IFN-γ). This increase in mRNA expression of IL-1ß, TNF-α, and IFN-γ was about 8.3, 10.6, and 4.5-fold, respectively, which significantly downregulated upon treatment. Collectively, P. americana is a promising medicinal plant with anticoccidial, antioxidant, and anti-inflammatory activities and could be used for the treatment of coccidiosis.

Effects of dietary sulfur amino acid levels on growth performance and intestinal immunity in broilers vaccinated and subsequently infected with coccidiosis.

Coccidia vaccination is a common practice in the poultry industry. However, research is lacking regarding the optimal nutritional support for coccidia vaccinated broilers. In this study, broilers were vaccinated with coccidia oocyst at hatch and were fed with a common starter diet from 1 to 10 d. On d 11, the broilers were randomly assigned to groups in a 4 × 2 factorial arrangement. Briefly, the broilers were fed one of four diets containing 0.6, 0.8, 0.9, and 1.0% of standardized ileal digestible methionine plus cysteine (SID M+C), respectively, from 11 to 21 d. On d 14, the broilers from each diet group were orally gavaged with either PBS (Mock challenge) or Eimeria oocysts. Compared to PBS-gavaged broilers and regardless of dietary SID M+C levels, the Eimeria-gavaged broilers had 1) decreased gain-to-feed ratio (15-21 d, P = 0.002; 11-21 d, P = 0.011); 2) increased fecal oocysts (P < 0.001); 3) increased plasma anti-Eimeria IgY (P = 0.033); and 4) increased intestinal luminal interleukin-10 (IL-10; duodenum, P = 0.039; jejunum, P = 0.018) and gamma interferon (IFN-γ; duodenum, P < 0.001; jejunum, P = 0.017). Regardless of Eimeria gavage, broilers fed 0.6% SID M+C had decreased (P<0.001) body weight gain (15-21 and 11-21 d) and gain-to-feed ratio (11-14, 15-21, and 11-21 d) when compared to those fed ≥ 0.8% SID M+C. Eimeria challenge increased (P < 0.001) duodenum lesions when the broilers were fed with 0.6, 0.8, and 1.0% SID M+C, and increased (P = 0.014) mid-intestine lesions when the broilers were fed with 0.6 and 1.0% SID M+C. An interaction between the two experimental factors was detected on plasma anti-Eimeria IgY titers (P = 0.022), as coccidiosis challenge increased plasma anti-Eimeria IgY titers only when the broilers were fed with 0.9% SID M+C. In summary, the dietary SID M+C requirement for grower (11-21 d) broilers vaccinated with coccidiosis was ranged from 0.8 to 1.0% for optimal growth performance and intestinal immunity, regardless of coccidiosis challenge.

Biosynthesized selenium nanoparticles to rescue coccidiosis-mediated oxidative stress, apoptosis and inflammation in the jejunum of mice.

One of the most crucial approaches for treating human diseases, particularly parasite infections, is nanomedicine. One of the most significant protozoan diseases that impact farm and domestic animals is coccidiosis. While, amprolium is one of the traditional anticoccidial medication, the advent of drug-resistant strains of Eimeria necessitates the development of novel treatments. The goal of the current investigation was to determine whether biosynthesized selenium nanoparticles (Bio-SeNPs) using Azadirachta indica leaves extract might treat mice with Eimeria papillata infection in the jejunal tissue. Five groups of seven mice each were used, as follows: Group 1: Non-infected-non-treated (negative control). Group 2: Non-infected treated group with Bio-SeNPs (0.5 mg/kg of body weight). Groups 3-5 were orally inoculated with 1×103 sporulated oocysts of E. papillata. Group 3: Infected-non-treated (positive control). Group 4: Infected and treated group with Bio-SeNPs (0.5 mg/kg). Group 5: Infected and treated group with the Amprolium. Groups 4 and 5 daily received oral administration (for 5 days) of Bio-SeNPs and anticoccidial medication, respectively, after infection. Bio-SeNPs caused a considerable reduction in oocyst output in mice feces (97.21%). This was also accompanied by a significant reduction in the number of developmental parasitic stages in the jejunal tissues. Glutathione reduced (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) levels were dramatically reduced by the Eimeria parasite, whereas, nitric oxide (NO) and malonaldehyde (MDA) levels were markedly elevated. The amount of goblet cells and MUC2 gene expression were used as apoptotic indicators, and both were considerably downregulated by infection. However, infection markedly increased the expression of inflammatory cytokines (IL-6 and TNF-α) and the apoptotic genes (Caspase-3 and BCL2). Bio-SeNPs were administrated to mice to drastically lower body weight, oxidative stress, and inflammatory and apoptotic indicators in the jejunal tissue. Our research thus showed the involvement of Bio-SeNPs in protecting mice with E. papillata infections against jejunal damage.