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Avian coccidiosis, caused by Eimeria spp, is a devastating disease in laying hens. Previous studies have suggested that amino acids may be involved in Eimeria infection of broiler chickens. However, their metabolic features in laying hens, as well as the effect of multiple Eimeria species challenges on poultry hosts have not been elucidated yet. Here, a targeted metabolomics approach was employed to identify altered amino acid metabolism and mechanisms in laying hens with multiple Eimeria species challenges. Laying hens, Hy-Line W-36 aged 25 wk, were randomly assigned to a control group and groups inoculated with varying levels of mixed Eimeria species (E. maxima, E. tenella, and E. acervulina). Serum samples from each group were collected at 6 d and 14 d of postinoculation (6 and 14 DPI) for metabolite profiling. Metabolomic analysis revealed notable metabolic variations between control and infected groups, especially at 6 DPI stage. Varying levels of Eimeria dosages did not show a significant metabolic difference, and metabolites were sensitive to low-level infection. With statistical analysis, differentially expressed compounds (3-methylhistidine, alanine, aspartate, lysine, asparagine, methionine, ornithine, and tryptophan) were selected, and their metabolic network was identified by pathway enrichment analysis. In the network, the lysine biosynthesis pathway was upregulated, while the arginine and proline metabolic pathway was downregulated under infection. Other pathways showed complex patterns of metabolic relationships. Based on the results, biological implications of metabolic changes were elucidated and discussed. Last, the results were further confirmed with our previous study (phenotype and gene expression results) using the same set of samples. Our finding provides in-depth information on altered amino acid metabolism and mechanisms in laying hens upon multiple Eimeria species infection., Competing Interests: DISCLOSURES The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The purpose of the study was to investigate the effects of the reduced dietary calcium (Ca) and phosphorus (P) level and supplementation of 25-hydroxycholecalciferol (25-OHD 3 ) on the expression of vitamin D receptor (VDR) and antimicrobial peptides and gut microbiota of broiler chickens with/without Eimeria challenge. A total of 576 fourteen-day-old broiler chicks were randomly allocated according to a 2 × 2 × 2 factorial design with main effects including Eimeria challenging (125,000 Eimeria acervulina, 25,000 Eimeria maxima, and 25,000 Eimeria tenella), dietary Ca and P levels (0.84% Ca and 0.42% available P or 0.64% Ca and 0.22% available P), and supplementation of 25-OHD 3 (3,000 IU/kg) of 6 replicates. Three-way ANOVA was performed, and the effects of 3 main factors and their interactions were investigated. The reduced dietary Ca and P level downregulated cathelicidins 3 (CATH3) in the upper jejunum in the Eimeria challenging condition (interaction; P < 0.05). The reduced dietary Ca and P level decreased the relative mRNA expression of jejunal avian beta defensin 5 (AvBD5) in the Eimeria challenging condition (interaction; P < 0.05). The reduced dietary Ca and P level tended to decrease the relative mRNA expression of jejunal AvBD9 in the Eimeria challenging condition (interaction; P = 0.051). The reduced dietary Ca and P level decreased observed features (alpha diversity parameter for richness) in the upper jejunal microbiota in the Eimeria challenging condition (interaction; P < 0.05). The supplementation of 25-OHD 3 decreased the relative abundance of the phylum Bacteroidetes (P < 0.05) and increased the relative abundance of the family Ruminococcaceae (P < 0.05) in the cecal digesta. The supplementation of 25-OHD 3 decreased the serum endotoxin level in the Eimeria challenging condition (interaction; P < 0.05). Therefore, the reduced dietary Ca and P level modulated the upper jejunal microbiota via modulating the expression of antimicrobial peptides, and the supplementation of 25-OHD 3 favorably modulated the cecal microbiota in broiler chickens with/without Eimeria challenge., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study investigated the best oral delivery strategy (gavage or feed) for the B. subtilis expressing the chicken anti-microbial peptide cNK-2 (B. subtilis-cNK-2) in comparison to monensin, in chickens challenged with Eimeria acervulina (E. acervulina). A total of 120 broiler chickens were randomly allocated into 5 treatment groups in a completely randomized design: 1) uninfected chickens fed with basal diet (NC), 2) E. acervulina-infected chickens fed a basal diet (PC), 3) E. acervulina-infected chickens fed a basal diet supplemented with 90 mg monensin/kg feed (MO), 4) E. acervulina-infected chickens fed a basal diet and orally gavaged with B. subtilis-cNK-2 at 1 × 10 10 cfu/d (CNK-O), and 5) E. acervulina-infected chickens fed a basal diet mixed with B. subtilis-cNK-2 at 1 × 10 10 cfu/kg feed (CNK-F). The challenge consisted of 5,000 sporulated E. acervulina oocysts through oral gavage on d 15. Body weights were measured on d 7, 14, 21, and 23. Duodenal tissue and digesta samples were collected at 6 d postinfection (dpi) to assess the gut integrity, oxidative stress, mucosal immunity, and the gut microbiome. Fecal samples were collected from 6 to 8 dpi to enumerate the oocyst shedding. Chickens in the CNK-O group showed improved (P < 0.05) growth performance, gut integrity, and mucosal immunity compared to PC, comparable to chickens in the MO group. Chickens in the MO, CNK-F, and CNK-O treatment groups all showed lower (P < 0.05) oocyst shedding compared to PC chickens. Moreover, distinct cytokine profile, oxidative stress measures, tight junction proteins, and shifts in the gut microbiome with associated functional changes were observed in all challenge groups. In conclusion, we showed that the oral administration of B. subtilis-cNK-2 improved growth performance, enhanced local protective immunity, and reduced fecal oocyst shedding in broiler chickens infected with E. acervulina, demonstrating potential use of B. subtilis-cNK-2 as an alternative to antibiotics to protect chickens against coccidiosis., Competing Interests: DISCLOSURES SSW, IP, YL, CGG, and HSL declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. JGVO, LMR and CP are stockholders and employees of US Biologic., (Published by Elsevier Inc.)

Mycotoxins, unavoidable contaminants in feed and feed ingredients, have the potential to influence the incidence and severity of various diseases upon ingestion. Sheep coccidiosis is an enteric disease caused by protozoa of Eimeria spp. However, the extent to which the presence of aflatoxin b1 (AFB1) synergistically exacerbates damage to intestinal health in lambs with Eimeria remains unclear. 50-day-old female lambs were randomly assigned to a 2 × 2 factorial arrangement of treatments for 15 days to assess the impact of AFB1 exposure on lambs with or without Eimeria (E.) ovinoidalis infection. Our findings reveal that AFB1 synergistically intensifies damage to intestinal health in lambs challenged by E. ovinoidalis. This is evidenced by disruptions to the intestinal microbiota and reductions in the production of short-chain fatty acids. AFB1 further aggravates damage to the cecal mechanical barrier. Additionally, AFB1 contributes to the entry of lipopolysaccharide into the bloodstream, activating the inflammatory response. Interestingly, AFB1 exposure history results in an early peak of oocyst excretion and a decreased number of oocyst excretion in E. ovinoidalis infected lambs. This may be closely linked to the destruction of the intestinal epithelial cell structure and its apoptosis, as indicated by a decreased ratio of Bcl-2 to Bax and increased caspase-3 levels. Mechanistically, proteomics analysis identified mitochondrial dysfunction (inhibition of the oxidative phosphorylation pathway) as the primary factor intensifying intestinal epithelial cell destruction caused by coccidia, exacerbated by AFB1 through the inhibiting the conversion of NADH to NAD+ in the cecum of lambs via down-regulation of the PGC-1α/NRF1/TFAM pathway. Overall, these results offer novel insights into the AFB1 complicity in accelerating intestinal damage caused by E. ovinoidalis in lambs. Targeting the mitochondrial oxidative phosphorylation pathway of the intestine may represent a new therapeutic strategy against the detrimental effects of mycotoxin and coccidia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Eimeria maxima is a major cause of coccidiosis in chickens and a key predisposing factor for other economically significant diseases such as necrotic enteritis. However, a detailed understanding of the intestinal microbiome response to E. maxima infection is still lacking. This study aimed to comprehensively investigate the dynamic changes of the intestinal microbiome for 14 days post-infection (dpi) with E. maxima . Bacterial 16S rRNA gene sequencing was performed with the ileal and cecal digesta collected from mock and E. maxima- infected chickens at the prepatent (3 dpi), acute (5 and 7 dpi), and recovery phases (10 and 14 dpi) of infection. Although no notable changes were observed at 3 dpi, significant alterations of the microbiota occurred in both the ileum and cecum at 5 and 7 dpi. By 14 dpi, the intestinal microbiota tended to return to a healthy state. Notably, Lactobacillus was enriched in response to E. maxima infection in both the ileum and cecum, although individual Lactobacillus , Ligilactobacillus , and Limosilactobacillus species varied in the temporal pattern of response. Concurrently, major short-chain fatty acid-producing bacteria, such as Faecalibacterium , were progressively suppressed by E. maxima in the cecum. On the other hand, opportunistic pathogens such as Escherichia , Enterococcus , and Staphylococcus were significantly enriched in the ileum during acute infection., Importance: We have observed for the first time the dynamic response of the intestinal microbiota to Eimeria maxima infection, synchronized with its life cycle. Minimal changes occur in both the ileal and cecal microbiota during early infection, while significant alterations coincide with acute infection and disruption of the intestinal mucosal lining. As animals recover from coccidiosis, the intestinal microbiota largely returns to normal. E. maxima -induced intestinal inflammation likely creates an environment conducive to the growth of aerotolerant anaerobes such as Lactobacillus , as well as facultative anaerobes such as Escherichia , Enterococcus , and Staphylococcus , while suppressing the growth of obligate anaerobes such as short-chain fatty acid-producing bacteria. These findings expand our understanding of the temporal dynamics of the microbiota structure during Eimeria infection and offer insights into the pathogenesis of coccidiosis, supporting the rationale for microbiome-based strategies in the control and prevention of this condition., Competing Interests: The authors declare no conflict of interest.

Many plants are efficient anticoccidial agents owing to their content of active chemicals. Drug-resistant Eimeria species have emerged as a result of excessive drug use. The current work aimed to investigate the oocysticidal activity (Eimeria papillata) of Olea europaea stem extract (OESE) and leaf extract (OELE) in vitro. The results of gas chromatography-mass spectrometry analysis for OELE and OESE showed the presence of 12 and 9 phytochemical compounds, respectively. Also, chemical examination revealed that the plant extracts are rich in phenols, flavonoids and tannins. Additionally, the best radical scavenging activity of OESE and OELE was at a concentration of 100 μg/ml, reaching 92.04 ± 0.02 and 92.4 ± 0.2%, respectively. The in vitro study revealed that concentrations of 200 mg/ml from OESE and OELE caused significant inhibition (100%) of process sporulation for E. papillata oocysts, in contrast to the other commercial products, which displayed varying degrees of suppression sporulation. Our findings showed that OESE and OELE have anticoccidial activity, which motivates further the conduction of in vivo studies in the search for a less expensive and more efficient cure., (© 2024 John Wiley & Sons Ltd.)

The health and productivity of broilers may be improved by optimizing the availability and levels of trace minerals (TM) in their feed, especially in the presence of parasites. This study investigated the effects of replacing inorganic TM (ITM) with an advanced chelate technology-based 7 TM (ACTM) on performance, hematology, lesion score, oocyst shedding, gut morphology, and tight junction structure in broilers challenged with mixed Eimeria species. There were 480 1-day-old broiler chickens divided into 5 groups: uninfected negative control and recommended levels of ITM (NC); infected positive control and recommended levels of ITM (PC); or PC supplemented with salinomycin (SAL); PC diet with 50 % ACTM instead of ITM (ACTM50); or PC diet with 100 % ACTM instead of ITM (ACTM100). All groups, except NC, were orally challenged with mixed Eimeria spp. oocysts on day 14. Each group had 6 replicate cages, with 16 birds per replicate. The results showed that the NC, SAL, and ACTM100 groups had higher (P < 0.05) body weight, average daily gain (ADG), and European production efficiency index (EPEI), as well as a lower (P < 0.05) feed conversion, mortality rate, and heterophile to lymphocyte ratio compared to the PC group, with the NC group having the highest ADG and EPEI throughout the experiment. The SAL and ACTM100 groups had lower (P < 0.05) intestinal lesion scores and oocyst numbers compared to the PC group, although all coccidiosis-challenged groups had higher oocyst shedding compared to the NC group. On day 24, the challenged birds in the SAL and ACTM100 groups had higher (P < 0.05) villus height and surface area in the duodenum and ileum, as well as a higher (P < 0.05) villus height to crypt depth ratio in the jejunum. The expression levels of jejunal CLDN1 and ZO-1 were also higher (P < 0.05) in the ACTM100 and SAL groups compared to the PC and ACTM50 groups at 24 days of age. In conclusion, while using ACTM in broiler diets at 50 % of the commercial recommended levels maintained performance and physiological responses, complete replacement with ACTM improved growth performance and intestinal health characteristics, similar to salinomycin under Eimeria challenge conditions., Competing Interests: Declaration of Competing Interest Bonzachicken is designed and synthesized using the Advanced Chelate Compounds Technology, invented by Mr. Mohammad Hassan Nazaran and patented in the United States Patent and Trademark Office (US8288587B2). The authors of this research have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Live anticoccidial vaccines, either formulated with unattenuated or attenuated Eimeria parasites, are powerful stimulators of chicken intestinal immunity. Little is known about the dynamics of gene expression and the corresponding biological processes of chicken responses against infection with precocious line (PL) of Eimeria parasites. In the present study, we performed a time-series transcriptomic analysis of chicken duodenum across 15 time points from 6 to 156 hours post-infection (p.i.) with PL of E. acervulina. A high-quality profile showing two distinct changes in chicken duodenum mRNA expression was generated during the infection of Eimeria. Early response revealed that activation of the chicken immune response was detectable from 6 h.p.i., prominent genes triggered during the initiation of asexual and sexual parasite growth encompass immune regulatory effects, such as interferon gamma (IFN-γ), interferon regulatory factor 1 (IRF1), and interleukin-10 (IL10). The late response was identified significantly associating with maintaining cellular structure and activating lipid metabolic pathways. These analyses provide a detailed depiction of the biological response landscape in chickens infected by the PL of E. acervulina, contributing significant insights for the investigation of the host-parasite interactions and the management of parasitic diseases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: A variety of bioactive compounds isolated from various botanical sources have been found to have therapeutic and immunotherapeutic effects on chicken coccidiosis., Aim: This study aimed to evaluate the anti-coccidial potential of Enteromorpha prolifera polysaccharide (EP) in indigenous chickens in Northwest Ethiopia., Materials and Methods: A total of 78 male indigenous chickens were used for this study. The study had two treatment groups: (1) the EP non-supplemented group (those fed on diets without EP and Eimeria oocyst inoculated) and (2) the EP group (those receiving diets supplemented with 400 mg EP/kg diet and Eimeria oocyst inoculated). Each treatment group had five replications. Following fourteen days of EP supplementation, 1.5 × 10 4 oocysts of mixed Eimeria species were inoculated into individual birds., Results: EP-supplemented chicken showed significantly lower (p < 0.05) oocyst counts compared to non-supplemented ones on 9 and 11 days post-challenge. In addition, chickens in the EP-supplemented group showed less severe lesion scores, with an average score of 1.33. Chickens that received EP showed a maximum of 27.27% protection against lesions. In contrast, the non-supplemented chickens had a lower percentage of protection (19.83%). The maximum anti-coccidial index value (146.98) was obtained from EP-supplemented chickens. Chickens in the EP-supplemented group exhibited a significantly higher (p < 0.05) weight gain., Conclusion: Overall, the inclusion of EP in chickens' diets shows promise as a potential anti-coccidial strategy. However, additional research is required to explore the mechanisms by which EP in chickens' diet could involve in increasing the protection ability of chickens against coccidiosis., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Strategies to counteract interleukin (IL)-10-mediated immune evasion by Eimeria spp. during coccidiosis- like anti-IL-10 antibodies- may protect broiler chicken health and reduce incidence of secondary necrotic enteritis (Clostridium perfringens) via undetermined mechanisms. Objectives were to use sequencing techniques to evaluate jejunal microbial community composition and function in anti-IL-10-fed broilers during coccidiosis and necrotic enteritis. On d0, Ross 308 chicks were placed in 32 cages (15 chicks/ cage) for a 25-d study and randomly assigned to diets ± 0.03% anti-IL-10. Six chicks/ diet were euthanized for distal jejunum content and tissue collection on d 14 (baseline) before inoculating the remainder with saline or 15,000 E. maxima oocysts (M6 strain). Half the chicks challenged with E. maxima were challenged with C. perfringens (1×10 8 colony forming units) on d 18 and 19. Follow-up samples (6 chicks/treatment) were collected at 7 and 11 d postinoculation (pi) for the E. maxima-only group, or 3 and 7 dpi for the E. maxima + C. perfringens group with 3/7 dpi being designated as peak and 7/11dpi as postpeak challenge. DNA was extracted from digesta for microbiota composition analysis (16S rRNA gene sequencing) while RNA was extracted from tissue to evaluate the metatranscriptome (RNA sequencing). Alpha diversity and genus relative abundances were analyzed using the diet or challenge main effects with associated interactions (SAS 9.4; P ≤ 0.05). No baseline microbial changes were associated with dietary anti-IL-10. At peak challenge, a diet main effect reduced observed species 36.7% in chicks fed anti-IL-10 vs. control; however, the challenge effect reduced observed species and Shannon diversity 51.2-58.3% and 33.0 to 35.5%, respectively, in chicks challenged with E. maxima ± C. perfringens compared to their unchallenged counterparts (P ≤ 0.05). Low sequencing depth limited metatranscriptomic analysis of jejunal microbial function via RNA sequencing. This study demonstrates that challenge impacted the broiler distal jejunum microbiota more than anti-IL-10 while future research to characterize the microbial metatranscriptome may benefit from investigating other intestinal compartments., Competing Interests: DISCLOSURES The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Dietary anti-interleukin (IL)-10 antibodies may protect broiler performance during coccidiosis by inhibiting Eimeria host-evasion pathways; however, anti-IL-10's effects on microbial communities during coccidiosis and secondary Clostridium perfringens (necrotic enteritis) challenge is unknown. The study objectives were to assess the jejunal microbiota of broilers fed anti-IL-10 during E. maxima ± C. perfringens challenge. Two replicate studies using Ross 308 chicks placed in wire-floor cages (32 cages/ replicate study; 20 chicks/ cage) were conducted, with chicks assigned to diets ± 0.03% anti-IL-10 for 25 d. In both replicate studies, challenge-designated chicks were inoculated with 1 × 10 8 Salmonella Typhimurium colony forming units (CFU) at placement. On d14, S. Typhimurium-inoculated chicks were gavaged with 15,000 sporulated Eimeria maxima M6 oocysts and half the E. maxima-challenged chicks received 1×10 8 C. perfringens CFUs on d 18 and 19. Six chicks/ treatment were euthanized for distal jejunum content collection at baseline (d 14), 7 d post-inoculation (pi) with E. maxima/ 3 dpi with C. perfringens (peak) or 11 dpi with E. maxima/ 7 dpi with C. perfringens (post-peak) for 16S rRNA gene amplicon sequencing. Sequences were quality screened (Mothur V.1.43.0) and clustered into de novo operation taxonomical units (OTU; 99% similarity) using the SILVA reference database (v138). Alpha diversity and log-transformed relative abundance data were analyzed in SAS 9.4 with replicate study, diet, challenge, and timepoint main effects plus associated interactions (P ≤ 0.05). Few baseline changes were observed, but E. maxima ± C. perfringens challenge reduced Romboutsia and Staphylococcus relative abundance 4- to 800-fold in both replicate studies (P ≤ 0.008). At peak challenge with secondary C. perfringens, feeding anti-IL-10 instead of the control diet reduced Clostridium sensu stricto 1 relative abundance 13- and 1,848-fold in both replicate studies (P < 0.0001); however, OTUs identified as C. perfringens were not affected by dietary anti-IL-10. These results indicate that anti-IL-10 does not affect the jejunal microbiota of unchallenged broilers, while coccidiosis or necrotic enteritis challenge generally contributed to greater microbiota alterations than diet., Competing Interests: DISCLOSURES The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Vitamin D 3 (25-hydroxyvitamin D 3 (VD)) and vitamin E (VE) have proven to have immunomodulatory and antioxidant functions along with capacities to improve the reproductive function in chickens. Coccidiosis in laying hens at different stages of growth has been shown to negatively affect performance, immune response, and oxidative status, thus increasing the cost of production. A study was conducted to evaluate the influence of dietary VD or VE on performance, gut health, immune response, and oxidative status of laying hens at peak production. Laying hens (23 wk-of-age, n = 225) were randomly allocated into 5 treatment groups (n = 9 hens/replicate) with 5 replicate groups each: 1) unchallenged control (UC), 2) pair-fed control (PF), 3) challenged control (CC), 4) challenged control top-dressed with 5,000 IU of 25-hydroxyvitamin D 3 (VD) per kg of diet, and 5) challenged control top-dressed with 100 IU of DL-α-tocopherol (VE). At 25 wk-of-age, hens grouped in CC, VD, and VE were challenged with mixed Eimeria spp. to induce coccidiosis. VD or VE supplemented hens did not impact bird body weight; however, egg production increased by 10.36% and 13.77%, respectively (P < 0.0001). Furthermore, the gut health of the hens was improved with either VD or VE supplementation, as indicated by lowered gut permeability and intestinal lesion scores (P < 0.05). VE significantly reduced the heterophil count (P = 0.0490) alongside numerically increasing the peripheral CD4+ and CD8+ T cells and monocyte counts (P > 0.05). Both VD or VE increased the TAC at 14 DPI compared to UC (P<0.05). Preliminary findings suggest that dietary VD or VE supplementation has the potential to improve gut health, modulate the immune response, and increase egg production in coccidiosis-infected laying hens., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Necrotic enteritis (NE) is a disease of worldwide distribution, which affects young broilers and causes economic losses on a scale of 6 billion dollars per year. For decades, NE was controlled in poultry flocks by dietary administration of low doses of antimicrobial growth promoters (AGPs). However, an increase in NE incidence was noted after the AGP ban. This study aimed to compare the effect of an antibiotic (Enramycin) diet to a combination of sodium butyrate, hydrolyzed yeast, and zinc proteinate (Viligen TM ) on broiler diets regarding performance, blood parameters, intestinal permeability, morphology and lesions, and carcass yield of broilers challenged with Eimeria spp. and Clostridium perfringens to simulate subclinical necrotic enteritis. A total of 1,150 one-day-old male broiler chickens with an initial average weight of 43.9 ± 0.65 g were allocated to 50 experimental pens. Animals were divided into 5 groups: Negative control (NC) without additives; Positive control (PC) with 0.12 g/ton of Enramycin (8%); V500, V1000, and V1500 with the addition of 500, 1.000, and 1.500 g/ton of Viligen, respectively. All animals were challenged by Eimeria spp. at 7 d of age and by C. perfringens at 17, 18, and 19 d for induction of subclinical NE. The broilers fed with all concentrations of Viligen showed similar performance, blood parameters, intestinal permeability, and carcass yield compared to PC broilers. However, NC broilers showed higher FCR compared to PC broilers from 1 to 33 d (1.42 vs. 1.39) (P = 0.048) and from 1 to 42 d (1.51 vs. 1.49) (P < 0.001). V1500 broilers had fewer intestinal lesions at 28 d when compared to the PC treatment (P < 0.05) and showed that higher Viligen inclusion resulted in lower intestinal damage. At 21 d, the V500 group showed higher intestinal morphology characteristics (VH:VD 4.9 vs. 3.5) compared to the PC treatment (P < 0.001). Thus, in this study, the dietary addition of Viligen to broilers challenged by an experimental model of subclinical NE resulted in lower intestinal damage and similar performance to that obtained by the addition of Enramycin., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

The water can be a significant risk factor for the occurrence of cattle eimeriosis on farms located in tropical climate regions. This study aimed to assess the viability period of sporulated oocysts of bovine Eimeria spp. in water containing organic matter and to evaluate the efficacy of disinfectants against sporulated oocysts of this protozoa. Two experiments were conducted, each comprising in vitro and in vivo evaluations. In Experiment 1, the viability period of oocysts sporulated in buckets containing a solution composed of a mixture of feces with oocysts, water, and potassium dichromate was assessed. Quantifications and identification of Eimeria spp. oocysts were performed on days + 30, + 60, + 90, and every 30 days thereafter until no more oocysts were detected. Naïve calves were inoculated with this solution kept in the bucket for 6 and 12 months. In Experiment 2, the efficacy of various disinfectants (acetic acid, sodium hypochlorite, ethanol + acetic acid, and ammonia quaternary) against a new solution of sporulated oocysts and pH changes over time (10', 30', and 24 h) were evaluated in the laboratory. The most effective disinfectant was used to treat the water solution and inoculate the calves, and its efficacy was calculated. In Experiment 1, Eimeria spp. oocysts remained viable in the solution for up to 12 months. E. zuernii persisted until the end of the study period. Calves inoculated with the solution kept in the buckets for 6 and 12 months excreted at least four Eimeria species (E. zuernii, E. bovis, E. ellipsoidalis, and E. cylindrica). In Experiment 2, among the tested disinfectants, 3% acetic acid demonstrated the highest efficacy (10' = 80.9%; 30' = 87.9%; 24 h = 91.7%). The pH values of the solutions containing acetic acid ranged from 2.4 to 3.5. Calves that received the inoculum treated with 3% acetic acid did not excrete Eimeria spp. oocysts in feces during the study period (efficacy = 100%). In contrast, all animals in the control group excreted Eimeria spp. oocysts in feces. Future studies should be carried out to better understand the possible effects of cattle drinking water with 3% acetic acid., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Early nutritional management approach greatly impacts broilers' performance and resistance against coccidiosis. The current study explored the impact of post-hatch feeding with a combination of glutamine (Glut) and different levels of omega-3 on broiler chickens' growth performance, muscle building, intestinal barrier, antioxidant ability and protection against avian coccidiosis. A total of six hundred Cobb 500 was divided into six groups: first group (fed basal diet and unchallenged (control) and challenged (negative control, NC) groups were fed a basal diet without additives, and the other groups were infected with Eimeria spp and supplemented with 1.5% Glut alone or with three different levels of omega-3 (0.25, 0.5 and 1%) during the starter period. Notable improvement in body weight gain was observed in the group which fed basal diet supplemented with glut and 1% omega 3 even after coccidia infection (increased by 25% compared challenged group) while feed conversion ratio was restored to control. Myogeneis was enhanced in the group supplemented with Glut and omega-3 (upregulation of myogenin, MyoD, mechanistic target of rapamycin kinase and insulin like growth factor-1 and downregulating of myostatin genes). Groups supplemented with Glut and higher levels of omega-3 highly expressed occluding, mucin-2, junctional Adhesion Molecule 2, b-defensin-1 and cathelicidins-2 genes. Group fed 1% Glut + omega-3 showed an increased total antioxidant capacity and glutathione peroxidase and super oxide dismutase enzymes activities with reduced levels of malondialdehyde, reactive oxygen species and H 2 O 2 . Post-infection, dietary Glut and 1% omega-3 increased intestinal interleukin-10 (IL) and secretory immunoglobulin-A and serum lysozyme, while decreased the elevated inflammatory mediators comprising interleukin IL-6, tumor necrosis factor-alpha, nitric oxide (NO) and inducible NO synthase. Fecal oocyst excretion and lesions score severity were lowered in the group fed 1% Glut and omega 3. Based on these findings, dietary Glut and omega-3 supplementation augmented restored overall broilers' performance after coccidial challenge.

The apicomplexan Eimeria ovinoidalis is distributed worldwide. It can cause clinical coccidiosis, which is one of the most pathogenic species in sheep, reducing growth rates and resulting in significant economic losses in the industry. Its principal clinical sign is profuse diarrhoea in young animals. In this study, we established a model of E. ovinoidalis infection in lambs to understand its pathogenicity and evaluate the gut microbiota and fecal metabolite profiles. Specifically, we observed a significant shift in the abundance of bacteria and disrupted metabolism in lambs. Especially during the peak period of excrete oocysts, it promoted the reproduction of some harmful bacteria in Proteobacteria and Actinobacteriota, and reduced the abundance of beneficial bacteria such as Lachnospiraceae and Rikenellaceae. In the later stage of the patent period, the abundance of harmful bacteria in the intestine decreased, the abundance of beneficial bacteria which could produce anti-inflammatory substances began to increase, and the abundance and diversity of intestinal flora also tended to parallel with the control group. Coccidia infection could lead to the increase of differential metabolites and metabolic pathways between infected and control group, but the difference decreased with time. During the peak period of excrete oocysts, although the antimicrobial metabolites such as Lividamine were up-regulated, the excess of these metabolites could still induce the production of endotoxin, while Butanoic acid and other anti-inflammatory metabolites decreased significantly. A metabolomics analysis showed that E. ovinoidalis infection altered metabolites and metabolic pathways, with biosynthesis of unsaturated fatty acids, Teichoic acid biosynthesis and Butanoate metabolism as the major disrupted metabolic pathways. Details of the gut microbiota and the metabolome after infection with E. ovinoidalis may aid in the discovery of specific diagnostic markers and help us understand the changes in parasite metabolic pathways., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In the first study, an in vitro culture system was developed to investigate the effects of carnosine on macrophage proinflammatory cytokine response using an established chicken macrophage cell line (CMC), gut integrity using a chicken intestinal epithelial cell line (IEC), muscle differentiation in quail muscle cells (QMCs) and primary chicken embryonic muscle cells (PMCs), and direct anti-parasitic effect against Eimeria maxima sporozoites. Cells to be tested were seeded in 24-well plates and treated with carnosine at 4 different concentrations (0.1, 1.0, and 10.0 µg). After 18 h of incubation, cells were harvested to measure gene expression of proinflammatory cytokines in CMC, tight junction (TJ) proteins in IECs, and muscle cell growth markers in QMCs and PMCs. In vivo trials were conducted to investigate the effect of dietary carnosine on disease parameters in broiler chickens challenged with E. maxima. One hundred and twenty male broiler chickens (0-day-old) were allocated into 4 treatment groups: 1) basal diet without infection (NC), 2) basal diet with E. maxima infection (PC), 3) carnosine at 10.0 mg/kg feed with PC (HCS), and 4) carnosine at 1.0 mg/kg feed with PC (LCS). All groups except NC were orally infected with E. maxima on d 14. Jejunal samples were collected for lesion scoring and jejunum gut tissues were used for transcriptomic analysis of cytokines and TJ proteins. In vitro, carnosine treatment significantly decreased IL-1β gene expression in CMC following LPS stimulation. In vivo feeding studies showed that dietary carnosine increased BW and ADG of chickens in E. maxima-infected groups and reduced the jejunal lesion score and fecal oocyst shedding in HCS group. Jejunal IL-1β, IL-8, and IFN-γ expression were suppressed in the HCS group compared to PC. The expression levels of claudin-1 and occludin in IECs were also increased in HCS following carnosine treatment. In conclusion, these findings highlight the beneficial effects of dietary carnosine supplementation on intestinal immune responses and gut barrier function in broiler chickens exposed to E. maxima infection., Competing Interests: DISCLOSURES Author AHS and TGR were employed by the Arm & Hammer Animal and Food Production. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Published by Elsevier Inc.)

The objective of this study was to evaluate the effect of 25% arginine supplementation as a functional amino acid in partially alleviating the detrimental effects of necrotic enteritis (NE) on the growth performance, serum biochemistry, gut integrity, and the relative gene expression of tight junction proteins and inflammatory cytokines in broilers during NE. Three hundred and sixty 1-day-old chicks were randomly allocated to 4 treatments in a 2 × 2 factorial arrangement -basal diet and 125% arginine diet, with or without NE challenge. NE was induced by inoculating 1 × 10 4 Eimeria maxima sporulated oocysts on d 14 and 1 × 10 8 CFU/bird C. perfringens on d 19, 20, and 21. The NE challenge had a significant effect on the BWG (p < 0.05), FCR (p < 0.05), serum AST (p < 0.05), GLU (p < 0.05), and K + (p < 0.05) levels, and intestinal permeability (p < 0.05) and jejunal lesion score (p < 0.05). A significant challenge × diet interaction effect was observed in the cecal tonsil CD8 + : CD4 + T-cell ratio on d 21 (p < 0.05) and 28 (p < 0.05) and spleen CD8 + : CD4 + T-cell ratio on d 21 (p < 0.05) and 35 (p < 0.05). Arginine supplementation significantly increased the CD8 + : CD4 + T-cell ratio in uninfected birds but decreased the CD8 + : CD4 + T-cell ratio in infected birds. On d 21, a significant interaction effect was observed on the relative expression of the iNOS gene (p < 0.05). Arginine supplementation significantly downregulated the expression of the iNOS gene in infected birds. A significant effect of the challenge (p < 0.05) was observed on the relative gene expression of the ZO-1 gene in the jejunum. NE challenge significantly downregulated the expression of the ZO-1 gene on d 21. In conclusion, arginine supplementation did not alleviate the depression in growth performance and disease severity during the NE challenge. However, arginine downregulated the expression of inflammatory cytokines and enzymes, preventing inflammatory injury to the tissues during NE. Hence, arginine might be supplemented with other alternatives to downregulate inflammatory response during NE in poultry., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A 35-d study investigated the impact of dietary supplementation with Arginine (Arg) or branched-chain amino acids (BCAA) of broilers receiving low-protein diets whilst infected with mixed Eimeria species. All birds were given the same starter (d0-10) and finisher (d28-35) diets. The 4 grower diets used were a positive control (PC) with adequate protein (18.5%), a low protein diet (NC;16.5% CP), or the NC supplemented with Arg or BCAA. Supplemental AA was added at 50% above the recommended levels. The treatments were in a 4 × 2 factorial arrangement, with 4 diets, with or without Eimeria inoculation on d14. Birds and feed were weighed after inoculation in phases: prepatent (d14-17), acute (d18-21), recovery (d22-28), and compensatory (d29-35). Ileal digesta, jejunum, and breast tissue were collected on d21, 28, and 35. There was no diet × Eimeria inoculation on growth performance at any phase. Infected birds weighed less and consumed less feed (P < 0.05) in all phases. In the prepatent and acute phases, birds on the Arg diets had higher weight gain (P < 0.05) and lower FCR, similar to PC, when compared to NC and BCAA-fed ones. Infection reduced AA digestibility on d21 and 28 (Met and Cys). However, birds that received supplemental AA had higher digestibility (P < 0.05) of their respective supplemented AA on d 21 only. Infected birds had lower (P < 0.05) BO + AT and higher PEPT1 expression on d21. There was a diet × Eimeria interaction (P = 0.004) on gene expression at d28; 4EBP1 genes were significantly downwardly expressed (P < 0.05) in birds fed Arg diet, irrespective of infection. Infected birds exhibited an upward expression (P < 0.05) of Eef2 on d21 and d28 but experienced a downward expression on d35. Supplemental Arg and BCAA had variable effects on growth performance, apparent ileal AA digestibility, and genes of protein synthesis and degradation, but the effect of Arg on promoting weight gain, irrespective of the Eimeria challenge, was more consistent., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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 × 10 4 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., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The study examined the effects of successive feeding of sources of n-3 PUFA to broiler breeders (BB) and their progeny in broiler chickens challenged with Eimeria. The BB were fed: 1) control (CON), corn-soybean meal diet, 2) CON + 1 % microalgae (DMA), as a source of DHA and 3) CON + 2.50% co-extruded full fat flaxseed (FFF), as a source of ALA. Eggs were hatched at 34, 44, and 54 wk of age. Posthatch treatments (BB-progeny) were: CON-CON, DMA-CON, FFF-CON, DMA-DMA and FFF-FFF with diets formulated for starter (d 1-10) and grower/finisher (d 11-42) phases. All chicks were orally challenged with Eimeria (E. acervulina and E. maxima) on d 10. Relative to CON, DMA and FFF increased concentration of n-3 PUFA by ≥ 2-fold in hatching eggs and progeny diets. There were no (P > 0.05) interactions between treatment and BB age on d 0 to 10 growth. In general, BB age affected (P < 0.05) growth performance throughout the study. In the starter phase, successive exposure to DHA and ALA improved FCR over CON-CON (P < 0.01). The interaction between treatment and BB age in grower/finisher was such that DHA exposure to younger BB resulted in poor growth performance (P < 0.05) relative to exposure to older BB. In contrast, exposure to ALA had similar (P > 0.05) growth performance irrespective of BB age. Moreover, successive exposure to ALA resulted in higher BWG, breast weight and lower FCR compared to successive exposure to DHA (P < 0.05). There were no (P > 0.05) interactions between treatment and BB age on the intestinal lesion scores, lymphoid organ weights and concentration of plasma immunoglobulin A (IgA). Successive exposure to DHA resulted in higher (P = 0.006) jejunal lesion scores than CON-CON birds. The results showed that successive exposure of DHA and ALA improved FCR relative to non-exposed birds in the starter phase. However, responses in the grower/finisher phase depended on n-3 PUFA type, with birds on successive ALA exposure supporting better growth and breast yield than birds on successive DHA exposure., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Eimeria is a protozoan parasite that affects poultry, particularly chickens, causing a disease known as coccidiosis. This disease imposes substantial significant economic challenges to the poultry sector., Objectives: The current study aimed to estimate the global prevalence and associated risk factors of Eimeria in domestic chickens., Methods: Multiple databases (Scopus, PubMed, ProQuest, Web of Science and Google Scholar) were searched for articles published until June 2023. The pooled prevalence was estimated using a random-effects model with a 95% confidence interval. The statistical analysis was conducted using meta packages in R version (3.6.1)., Results: In total, 41 articles fulfilled the eligibility criteria. The global pooled prevalence was 44.3% (36.9%-51.8%) with Eimeria tenella (38.7%, 30.1%-47.7%) as the most prevalent species. The highest pooled prevalence was related to the Western Pacific Region (80.5%, 72.6%-87.3%) and urban areas (44.4%, 36.5%-52.6%). Moreover, areas with humid subtropical climates represent the highest overall prevalence (75.8%, 46.6%-95.9%)., Conclusion: The necessity for robust and innovative strategies for preventing and managing this disease cannot be overstated. Addressing Eimeria impact is crucial not only for safeguarding poultry health but also for sustaining the economic viability of the poultry industry., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

The objective of this study was to evaluate the effects of 25% and 35% arginine supplementation in partially alleviating the effects of necrotic enteritis (NE) challenge on the production performance, intestinal integrity, and relative gene expression of tight junction proteins and inflammatory cytokines in broilers. Four hundred and eighty 1-day-old chicks were randomly allocated to the 4 treatments- Uninfected + Basal, NE + Basal, NE + Arg 125%, and NE + Arg 135%. NE was induced by inoculating 1 × 10 4 Eimeria maxima sporulated oocysts on d 14 and 1 × 10 8 CFU/bird C. perfringens on d 19, 20, and 21 of age by oral gavage. The NE challenge significantly decreased body weight gain (BWG) (p < 0.05) and increased the feed conversion ratio (FCR) (p < 0.05). On d 21, the NE challenge also increased the jejunal lesion score (p < 0.05) and relative gene expression of IL-10 and decreased the expression of the tight junction proteins occludin (p < 0.05) and claudin-4 (p < 0.05). The 125% arginine diet significantly increased intestinal permeability (p < 0.05) and the relative gene expression of iNOS (p < 0.05) and IFN-γ (p < 0.05) on d 21 and the bile anti-C. perfringens IgA concentration by 39.74% (p < 0.05) on d 28. The 135% arginine diet significantly increased the feed intake during d 0 - 28 (p < 0.05) and 0 to 35 (p < 0.05) and increased the FCR on d 0 to 35 (p < 0.05). The 135% and 125% arginine diet increased the spleen CD8 + : CD4 + T-cell ratio on d 28 (p < 0.05) and 35 (p < 0.05), respectively. The 135% arginine diet increased the CT CD8 + :CD4 + T-cell ratio on d 35 (p < 0.05). In conclusion, the 125% and 135% arginine diets did not reverse the effect of the NE challenge on the growth performance. However, the 125% arginine diet significantly increased the cellular and humoral immune response to the challenge. Hence, the 125% arginine diet could be used with other feed additives to improve the immune response of the broilers during the NE challenge., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Coccidiosis caused by Eimeria spp. results in substantial economic losses in the poultry industry. The objective of this study was to investigate the effects of dietary supplementation with n-3 polyunsaturated fatty acids-enriched fish oil on growth performance, intestinal barrier integrity, and intestinal immune response of broilers challenged with Eimeria spp. A total of 576 fourteen-day-old broilers were randomly assigned in a completely randomized design with a 3 × 2 factorial arrangement, comprising 2 diets supplemented with either 5% fish oil or 5% soybean oil, and 3 Eimeria spp. infection levels: a nonchallenge control, a low dose of Eimeria challenge, and a high challenge dose. The results of the study revealed significant interactions between diet and Eimeria challenge to parameters of gut barrier integrity and feed intake. A significant interaction was observed in feed intake between 5 and 8 d postinfection (DPI), where the fish oil groups exhibited a higher amount of feed intake compared to the soybean oil diet groups after coccidiosis infection. The effects of the fish oil diet resulted in enhanced gut barrier integrity, as evidenced by a trend of decreased gastrointestinal leakage and a lower mean of small intestine lesion scores after Eimeria challenge. Additionally, significant interactions were noted between Eimeria spp. challenge and diet regarding jejunal crypt depth. The positive impact of the fish oil diet was particularly noticeable with the high Eimeria challenge dose. Overall, these findings underscore the relationship between the fish oil diet and Eimeria challenge on broiler chicken intestinal health. Dietary supplementation of fish oil has the potential to maintain small intestine barrier integrity with severe Eimeria infection conditions., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Although anticoccidials effectively control coccidiosis, a needed reduction in the reliance on antimicrobials in animal production leads to the exploration of alternative compounds. The present study aimed to test five different dietary treatments to counteract the negative impact of coccidiosis on broiler chickens' health and performance. 1-day-old male Ross 308 broilers (n = 960) were randomly assigned to one of eight treatments, with six cages per treatment (20 birds/cage). To the diet of the broiler chickens of treatments (Trt) 1-5, a synbiotic was added from d0-10. From d10-28, birds of Trt1 and Trt2 were fed synbiotics, whereas birds of Trt3 were fed diets with glutamine, and birds of Trt4 and Trt5 were fed diets with a combination of β-glucans and betaine. From d28-35 onwards, birds of Trt1 were fed a diet with a synbiotic, whereas birds of Trt2-4 received diets with glutamine, and birds of Trt5 were fed a non-supplemented diet. Birds of the positive control group (PC; Trt6) were fed a standard diet supplemented with an anticoccidial (Decoquinate). The challenged negative control (NCchall; Trt7) and non-challenged negative control (NC) Trt8 were fed a standard diet without anticoccidial or other dietary treatment. At 7 days (d) of age, all birds were inoculated with 1 023, 115, and 512 sporulated oocysts of E. acervulina, E. maxima, and E. tenella, respectively, except for Trt8. Body weight gain (BWG), feed intake, and feed conversion ratio were assessed for each feeding phase (d0-10, d10-28 and d28-35) and overall experimental period (d0-35). Oocyst shedding, Eimeria lesion scores, cecal length, and relative weight were assessed at d13, d22, d28 and d35. Additionally, oocyst shedding was determined at d9 and d17. Litter quality was evaluated at d27 and d34, and footpad lesions at d34. During the starter (d0-10) and finisher (d28-35) periods, performance did not differ between the treatments. During the grower period (d10-28), Trt6 (PC) and Trt8 (NC) chickens had the highest BWG of all treatments (P < 0.001). Dietary treatment had no effect on litter quality and severity of footpad lesions. In the PC group (Trt6), low oocyst excretion and lesion scores were found. When comparing Trt1-5 with NCchall (Trt7), none of the treatments significantly reduced oocyst output or lesion scores. In conclusion, in this experiment, none of the dietary treatments performed similar or better compared to the PC group (Trt6) regarding performance or reducing Eimeria oocyst shedding or lesion scores., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A total of 576-day-old Ross 308 broilers chicks (male) were used to evaluate the effect of various levels of pistachio green hull aqueous extract (PHE) and Eimeria challenge on the growth performance, intestinal health and antioxidant capacity. During infection period (25-42 d), treatments included: 1) control + unchallenged (negative control, NC), 2) 200 ppm PHE + unchallenged, 3) 300 ppm PHE + unchallenged, 4) 400 ppm PHE + unchallenged, 5) control + challenged (positive control, PC), 6) 200 ppm PHE + challenged, 7) 300 ppm PHE + challenged and 8) 400 ppm PHE + challenged (with 6 replications for each treatment). The outcomes revealed that in the challenged birds, average body weight gain (ABW), daily weight gain (DWG), and feed conversion ratio (FCR) linearly improved with increasing the PHE levels (P < 0.05). Infected broilers had lower daily feed intake (DFI) compared to unchallenged birds (P < 0.05). Villus height (VH), villus height to crypt depth (VH: CD) ratio and villus surface area (VSA) reduced linearly (P < 0.05), while muscle layer thickness (MT) increased linearly in challenged birds (P < 0.05). The consumption of the PHE significantly reduced the excreta oocytes and duodenum and jejunum lesion scores in Eimeria-challenged broilers (P < 0.05). By increasing the PHE levels, total antioxidant capacity (TAC) and superoxide dismutase (SOD) levels increased (P < 0.05), while the Eimeria challenge reduced TAC, SOD, and glutathione peroxidase (GPx) levels (P <0.05). In general, the use of PHE in the broilers diet improved the antioxidant capacity, birds performance, but diminished the excreta oocytes and lesion scores with no negative effect on the intestinal morphology., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Coccidiosis, a protozoan disease that substantially impacts poultry production, is characterized by an intracellular parasite. The study utilized 48 one-day-old Horro chickens, randomly divided into the infected (I) and control (C) groups. The challenge group of chickens were administered Eimeria maxima oocysts via oral gavage at 21-days-old, and each chicken received 2 mL containing 7×10 4 sporulated oocysts. The total RNAs of chicken jejunum and cecum tissues were isolated from three samples, each from I and C groups. Our study aimed to understand the host immune-parasite interactions and compare immune response mRNA profiles in chicken jejunum and cecum tissues at 4 and 7 days postinfection with Eimeria maxima. The results showed that 823 up- and 737 down-regulated differentially expressed mRNAs (DEmRNAs) in jejunum at 4 d infection and control (J4I vs. J4C), and 710 up- and 368 down-regulated DEmRNAs in jejunum at 7 days infection and control (J7I vs. J7C) were identified. In addition, DEmRNAs in cecum tissue, 1424 up- and 1930 down-regulated genes in cecum at 4 days infection and control (C4I vs. C4C), and 77 up- and 191 down-regulated genes in cecum at 7 days infection and control (C7I vs. C7C) were detected. The crucial DEmRNAs, including SLC7A5, IL1R2, GLDC, ITGB6, ADAMTS4, IL1RAP, TNFRSF11B, IMPG2, WNT9A, and FOXF1, played pivotal roles in the immune response during Eimeria maxima infection of chicken jejunum. In addition, the potential detection of FSTL3, RBP7, CCL20, DPP4, PRKG2, TFPI2, and CDKN1A in the cecum during the host immune response against Eimeria maxima infection is particularly noteworthy. Furthermore, our functional enrichment analysis revealed the primary involvement of DEmRNAs in small molecule metabolic process, immune response function, inflammatory response, and toll-like receptor 10 signaling pathway in the jejunum at 4 and 7 days postinfection. Similarly, in the cecum, DEmRNAs at 4 and 7 days postinfection were enriched in processes related to oxidative stress response and immune responses. Our findings provide new insights and contribute significantly to the field of poultry production and parasitology., (Copyright © 2024. Published by Elsevier Inc.)

We investigated the effects of supplementing low protein diets with methionine (Met) or threonine (Thr) during a mixed Eimeria (consisting of E. acervulina, E. maxima and E. tenella) challenge in broilers. All birds were fed the same starter diet (d1-9) and finisher diet (d28-35) which met Cobb 500 nutrient specifications. Birds were allocated to 1 of 4 dietary treatments from d9 to 28: a standard protein diet (19% CP); a low protein diet (16% CP); or the low protein diet supplemented with Met or Thr at 50% above recommendations. On d14, half of the birds were challenged, and half of the birds were unchallenged. From d14 to 28, feed intake was recorded daily and BW every 3 or 4 d. Oocyst excretion was measured daily from d18 to 27. On d21 and 28, 3 birds per pen were euthanized to assess nutrient digestibility, cytokine expression and intestinal histology. During the acute stage of the challenge, challenged birds reduced ADFI and ADG (P < 0.05). In the pre-patent and recovery stages, birds given the 16% CP diets increased ADFI (P < 0.05), meanwhile there were no differences in ADG in these stages (P > 0.05). Nutrient digestibility was reduced in challenged birds in the acute stage (P < 0.05) but tended to be greater than in unchallenged birds during the recovery stage. There was no significant effect of diet on oocyst excretion or intestinal histology (P > 0.05). Interactions were observed between diet and challenge on IL-10 and IL-21 expression in the cecal tonsils during the acute stage of the challenge (P < 0.05), due to reduced IL-10 expression in challenged Thr birds and greater IL-21 expression in challenged Met birds. Supplementation with Thr or Met had limited effects on the outcomes of a mixed Eimeria challenge but provides benefits to the host by enhancing their immune response., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Coccidiosis, an intestinal disease caused by Eimeria parasites, is responsible for major losses in the poultry industry by impacting chicken health. The gut microbiota is associated with health factors, such as nutrient exchange and immune system modulation, requiring understanding on the effects of Eimeria infection on the gut microbiota. This study aimed to determine the effects of Eimeria acervulina infection on the luminal and mucosal microbiota of the cecum (CeL and CeM) and ileum (IlL and IlM) at multiple time points (days 3, 5, 7, 10, and 14) post-infection. E. acervulina infection decreased evenness in CeL microbiota at day 10, increased richness in CeM microbiota at day 3 before decreasing richness at day 14, and decreased richness in IlL microbiota from day 3 to 10. CeL, CeM, and IlL microbiota differed between infected and control birds based on beta diversity at varying time points. Infection reduced relative abundance of bacterial taxa and some predicted metabolic pathways known for short-chain fatty acid production in CeL, CeM, and IlL microbiota, but further understanding of metabolic function is required. Despite E. acervulina primarily targeting the duodenum, our findings demonstrate the infection can impact bacterial diversity and abundance in the cecal and ileal microbiota., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

This study compared 2 herbal anticoccidiosis drugs (water-soluble and feed-additive drugs) with monensin coccidiostat, toltrazuril (TTZ, anticoccidiosis drug), and Livacox Q (anticoccidiosis vaccine) in terms of their effects on the prevention and treatment of coccidiosis in broilers. In this study, 280 Ross 308 broiler chickens (a mix of both genders) were used in a completely randomized design with 7 treatments and 5 replications each including 8 chickens per replicate. On d 21 of rearing, all experimental groups, except for the negative control group (NC), were challenged with a mixed suspension of common strains of Eimeria, and the intended indices were assessed, including performance indices, number of oocysts per gram (OPG) of feces, intestinal injuries, and the total number of intestinal bacteria. In addition, the NC and the group receiving the monensin had greater body weight gain (BWG) (P < 0.05). At the end of week 6, the monensin group had the highest feed intake (FI), while the water soluble medicine treatment resulted in the lowest feed intake (P < 0.05). Regarding the lesion scores on day 28, the highest and lowest rates of jejunal injuries were observed in the positive control group (PC), the monensin and vaccine group respectively. The rate of oocysts excretion (oocysts per gram of feces = OPG) on different days was higher in the PC group, and the use of monensin could further reduce excretion compared to the other groups (P > 0.05). Based on a comparison of the population of lactic acid bacteria between the NC and both medicinal plant treated groups, the use of these products could increase the population of these types of bacteria. Moreover, the population of Escherichia coli was less considerable in the NC and herbal powder groups (P < 0.05). Overall, similar to commercial medicines, the herbal medicines used in this project can be effective in the prevention and treatment of coccidiosis and can improve profitability in broiler rearing centers by improving intestinal health., Competing Interests: DISCLOSURES The authors declare that they have no conflict of interest in this study., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Chickens have undergone genetic improvements in the past few decades to maximize growth efficiency. However, necrotic enteritis (NE), an enteric disease primarily caused by C. perfringens, remains a significant problem in poultry production. A study investigated the differences in intestinal health between the nonselected meat-type chicken Athens Canadian Random Bred (ACRB) and the modern meat-type Cobb 500 broilers (Cobb) when challenged with experimental NE. The study utilized a 2 × 3 factorial arrangement, consisting of two main effects of chicken strain and NE challenge model (nonchallenged control, NC; NE challenge with 2,500/12,500 Eimeria maxima oocysts + 1 × 10 9 C. perfringens, NE2.5/NE12.5). A total of 432 fourteen-day-old male ACRB and Cobb were used until 22 d (8 d postinoculation with E. maxima on d 14, dpi), and the chickens were euthanized on 6 and 8 dpi for the analysis. All data were statistically analyzed using a two-way ANOVA, and Student's t-test or Tukey's HSD test was applied when P < 0.05. The NE12.5 group showed significant decreases in growth performance and relative growth performance from d 14 to 20, regardless of chicken strain (P < 0.01). The ACRB group exhibited significant decreases in relative body weight and relative body weight gain compared to the Cobb group from d 14 to 22 (P < 0.01). On 6 and 8 dpi, both NE challenge groups showed significant decreases in intestinal villus height to crypt depth ratio, jejunal goblet cell count, and jejunal MUC2 and LEAP2 expression (P < 0.01). Additionally, the NE12.5 group had significantly higher intestinal NE lesion score, intestinal permeability, fecal E. maxima oocyst count, intestinal C. perfringens count, and jejunal IFNγ and CCL4 expression compared to the NC group (P < 0.05). In conclusion, NE negatively impacts growth performance and intestinal health in broilers, parameters regardless of the strain., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Galliformes have a global distribution and are bred by humans as domestic animals or game birds. The world's poultry population is increasing due to high consumer demand for poultry products. Due to the increasing growth of Galliformes breeding, this study aimed to investigate gastrointestinal parasites in the feces of some species of Galliformes . In the present study, 100 distinct fecal samples were collected from seven different Galliformes species, including rural chickens, peacocks, partridges, pheasants, turkeys, quails, and guinea fowls. Sampling of each bird was performed separately, and these birds were kept in cages. In order to identify gastrointestinal parasites, the samples were evaluated using the Clayton-Lane method, the Modified Ziehl-Neelsen, and the Trichrome staining methods. Among 100 birds sampled, 53% were positive for gastrointestinal parasites. Moreover, 30%, 18%, and 5% of birds were infected with Eimeria spp. , nematodes, and Giardia spp. , respectively. The highest rate of parasitic infestation in Galliformes was related to protozoan infections, and the highest rate of protozoan infection was related to Eimeria (30%). In addition, complex infestation was not detected. Considering that most parasitic infections have been detected in apparently healthy birds, it is recommended to observe hygiene to regularly disinfect cages and reduce the density of birds in the nests. Due to the zoonotic potential of Giardia and the contamination of some birds with this parasite, this issue should be considered by public health officials, bird sellers' markets, owners, and breeders of these birds., Competing Interests: The authors declare there is no conflict of interest.

Coccidiosis is a protozoan intestinal disease that reduces the production of the sheep industry and causes large economic losses for sheep. Although chemically synthesised drugs are routinely employed to treat coccidiosis in sheep, the anticoccidial drug resistance and drug residues in edible meat have prompted an urgent search for alternatives. Herein, the anticoccidial properties of diclazuril, a conventional anticoccidial drug, and Allium sativum, Houttuynia cordata and Portulaca oleracea were assessed. Forty 45-day-old lambs naturally infected with Eimeria spp. were selected and randomly divided into five groups. The results showed that the sheep treated for coccidiosis had considerably decreased average daily gain (ADG) during both administration and withdrawal of the drug compared to the control group. Furthermore, at days 14, 21, 28 and 35, respectively, the three herbs and diclazuril had similar anticoccidial effects, with lower oocysts per gram (OPG) than the control group. On day 78, OPG in the three herbal groups was significantly lower than in the diclazuril group. In addition, the abundance and composition of the gut microbiota were changed in sheep treated with the three herbs and diclazuril compared to the untreated sheep. Moreover, some intestinal microorganisms have a correlation with OPG and ADG when using Spearman correlation analysis. In summary, our results suggest that all three herbs produce anticoccidial effects similar to diclazuril and modulate the balance of gut microbiota in growing lambs.

Intraepithelial lymphocytes (IEL) reside in the epithelium at the interface between the contents of the intestinal lumen and the sterile environment of the lamina propria. Because of this strategic location, IEL play a crucial role in various immunological processes, ranging from pathogen control to tissue stability. In mice and humans, IEL exhibit high diversity, categorized into induced IEL (conventional CD4 and CD8αβ T cells) and natural IEL (TCRαβCD8αα, TCRγδ, and TCR neg IEL). In chickens, however, the subpopulations of IEL and their functions in enteric diseases remain unclear. Thus, we conducted this study to investigate the role of IEL populations during necrotic enteritis (NE) in chickens. At 14 days of age, sixty-three Specific-pathogen-free (SPF) birds were randomly assigned to three treatments: Control (sham challenge), Eimeria maxima challenge (EM), and Eimeria maxima + Clostridium Perfringens ( C. Perfringens ) co-challenge (EM/CP). The EM and EM/CP birds were infected with Eimeria maxima at day 14 of age, and EM/CP birds were additionally orally inoculated with C. perfringens at days 18 and 19 of age. Birds were weighed at days 18, 20, and 26 of age to assess body weight gain (BWG). At 20 days of age (1 day-post C. perfringens infection; dpi), and 26 days of age (7 dpi), 7 birds per treatment were euthanized, and jejunum was harvested for gross lesion scores, IEL isolation, and gene expression. The EM/CP birds exhibited subclinical NE disease, lower BWG and shorter colon length. The Most changes in the IEL populations were observed at 1 dpi. The EM/CP group showed substantial increases in the total number of natural IEL subsets, including TCRαβ + CD4 - CD8 - , TCRαβ + CD8αα + , TCRγδ + , TCR neg and innate CD8α (iCD8α) cells by at least two-fold. However, by 7 dpi, only the number of TCRαβ + CD4 - CD8 - and TCRαβ + CD8αα + IEL maintained their increase in the EM/CP group. The EM/CP group had significantly higher expression of proinflammatory cytokines (IL-1β and IFN-γ) and Osteopontin (OPN) in the jejunum at 1 dpi. These findings suggest that natural IEL with innate and innate-like functions might play a critical role in the host response during subclinical NE, potentially conferring protection against C. perfringens infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Majeed, Hamad, Shah, Bielke and Nazmi.)

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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Coccidiosis stands as a protozoan disease of notable economic impact, characterized by an intracellular parasite that exerts substantial influence over poultry production. This invasion disrupts the integrity of the enteric mucosa, leading to the emergence of severe lesions and diminishing the efficiency of feed utilization in chickens. MicroRNA (miRNA) are short, non-coding RNA molecules with approximately 21-24 nucleotides long in size that play essential roles in various infectious diseases and inflammatory responses. However, the miRNA's expression patterns and roles in the context of Eimeria maxima infection of chicken intestines remain unclear. miRNA sequencing was employed to assess the miRNA expression profile in chicken jejunum during E. maxima infection. In this study, we analyzed miRNA expression profiles related to the host's immune response in the chicken jejunum during E. maxima infection. At 4 days infection and control (J4I versus J4C), 21 differentially expressed miRNAs in the jejunum were identified, comprising 9 upregulated and 12 downregulated miRNAs. Furthermore, in the jejunum, at 7 days infection and control (J7I versus J7C) groups, a total of 35 significantly differentially expressed miRNAs were observed, with 13 upregulated and 22 downregulated miRNAs. The regulatory networks were constructed between differentially expressed miRNA and mRNAs to offer insight into the interaction mechanisms between chickens and E. maxima coccidian infection. Furthermore, within the comparison group, we obtained 946, 897, and 281 GO terms that exhibited significant enrichment associated with host immunity in the following scenarios, J4I vs. J4C, J7I vs. J7C, and J4I vs. J7I, respectively. The KEGG pathway analysis indicated notable enrichment of differentially expressed miRNAs in the jejunum, particularly in J4I vs. J4C; enriched pathways included metabolic pathways, endocytosis, MAPK signaling pathway, regulation of actin cytoskeleton, and cytokine-cytokine receptor interaction. Moreover, in J7I vs. J7C, the KEGG pathway was significantly enriched, including metabolic pathways, protein processing in the endoplasmic reticulum, ubiquitin-mediated proteolysis, and FoxO signaling pathway. A comprehensive understanding of the host genetic basis of resistance with a combination of time-dependent infection to the Eimeria parasite is crucial for pinpointing resistance biomarkers for poultry production., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jebessa, Bello, Guo, Tuli, Hanotte and Nie.)

This study investigated the growth performance, nutrient utilization, and intestinal health responses of Eimeria-challenged broiler chickens to dietary Spirulina (Arthrospira platensis). On day 1, birds were assigned to 2 diets supplemented with Spirulina (0 or 5 g/kg) in a randomized complete block design. The birds within each diet were divided into 2 Eimeria-challenge groups (challenge or no-challenge) and that resulted in a 2 × 2 factorial arrangement with 2 levels each of Spirulina and challenge on day 14. On day 15, the birds in the challenge or no-challenge groups were orally gavaged with a solution containing Eimeria oocysts or 1% PBS, respectively. Samples were collected on days 21 and 26 (6- and 11-d post-infection; dpi). Data collected from days 1 to 26 were analyzed using the MIXED procedure of SAS. Birds that were fed Spirulina-supplemented diets had increased (P < 0.05) BW gain, gain-to-feed ratio, and total tract retention nitrogen from days 14 to 21. The ileal villus perimeter and area, serum catalase, HMOX1 and SOD1 jejunal abundance were all increased (P < 0.05) in birds fed Spirulina-supplemented diets on day 21 (6 dpi). However, there was no effect on bone ash or oocyst count. From days 21 to 26, there was a tendency (P = 0.059) for a Spirulina × Challenge interaction on the BW gain of birds. Moreover, dietary Spirulina addition increased (P < 0.05) serum catalase, total antioxidant capacity, ileal villus perimeter, tibia bone ash, and the relative mRNA expression of HMOX1, SOD1, claudin 1, and TNFα in the jejunal mucosa of birds on day 26 (11 dpi). On both 6 and 11 dpi, the Eimeria challenge negatively (P < 0.05) impacted growth performance, gut morphology, and the relative mRNA expression of genes. Overall, assessing the impact of Spirulina in broilers revealed its positive antioxidant, immune-modulating, and health benefits. However, its dietary addition did not completely reverse the Eimeria-induced effects in these birds. Ultimately, this study outlines the positive properties of dietary Spirulina beyond its use in the diet of healthy broiler chickens., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science.)

The objective of the present studies was to evaluate muramidase (MUR) supplementation in broilers under Eimeria and/or Clostridium perfringens challenge. For this, 2 experiments were conducted. Experiment 1. A total of 256 one-day old male Cobb 500 chicks were placed in battery cages in a completely randomized design, with 5 treatment groups, 7 replicate cages per treatment and 8 birds per cage. The treatments were: nonchallenged control (NC), challenged control (CC), CC + MUR at 25,000 or 35,000 LSU(F)/kg, and CC + Enramycin at 10 ppm (positive control-PC). Challenge consisted of 15× the recommended dose of coccidiosis vaccine at placement, and Clostridium perfringens (10 8 CFU/bird) inoculation at 10, 11, and 12 d. Macro and microscopic evaluation, immunohistochemistry, and gene expression were evaluated at 7, 14, 21, and 28 d of age. Experiment 2. A total of 1,120 one-day old male Cobb 500 chicks were placed in floor pens with fresh litter in a completely randomized design, with 4 treatment groups, 8 replicate pens per treatment, and 35 birds per pen. The treatments were: Control, supplementation of MUR at 25,000 or 45,000 LSU(F)/kg, and a positive control (basal diet plus Enramycin). At 10, 11, and 12 d of the experiment all the birds were inoculated by oral gavage with a fresh broth culture of a field isolate Clostridium perfringens (0.5 mL containing 10 6 CFU/bird). It was observed that in Experiment 1 MUR supplementation reduced the infiltration of macrophages and CD8+ lymphocytes in the liver and ileum of infected birds, downregulated IL-8 and upregulated IL-10 expression. In Experiment 2, MUR linearly improved the growth performance of the birds, increased breast meat yield, and improved absorption capacity. MUR supplementation elicited an anti-inflammatory response in birds undergoing a NE challenge model that may explain the improved growth performance of supplemented birds., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Eimeria necatrix is a high pathogenic pathogen, which seriously endangers the poultry industry. The surface antigens (SAGs) of Apicomplexa are a kind of membrane protein anchored on the surface of the parasites through its carboxyl terminal glycosylphosphatidylinositol (GPI) structure. However, little is known about GPI-linked surface proteins in E. necatrix. In the present work, the E. necatrix sag gene (Ensag -CAP ) was amplified and cloned for expression of the recombinant protein (rEnSAG -CAP ). The full length Ensag -CAP gene was 813 bp, coding 270 amino acids with a predicated molecular weight of 28.86 kDa and contained a CAP domain with four sequence motifs CAP1, CAP2, CAP3 and CAP4. The rEnSAG -CAP was about 32 kDa and mainly expressed in a soluble form. Western blot analysis indicated that the rEnSAG -CAP could be recognized by anti-rEnSAG -CAP monoclonal antibody (anti-rEnSAG -CAP McAb) and the convalescent serum of chicken infected with E. necatrix. Native protein of EnSAG -CAP was detected in second-generation merozoites (MZ-2) using anti-rEnSAG -CAP polyclonal antibody (anti-rEnSAG -CAP pAb). The findings from the indirect immunofluorescence assay and enzyme digestion utilizing Bacillus cereus phosphoinositide-specific phospholipase C (PI-PLC) revealed that EnSAG -CAP predominantly localized at the surfaces of SZ and MZ-2 via a GPI anchor. It was observed that EnSAG -CAP can be cleaved from MZ-2 by PI-PLC. Real-time quantitative PCR (qPCR) analysis showed that transcript levels of Ensag -CAP in MZ-2 was significantly higher than that in SZ (P < 0.05). The anti-rEnSAG -CAP McAb in vitro could significantly inhibit the sporozoite invasion into MDBK cells (P < 0.01), which suggests that the protein might participate in sporozoite invasion into MDBK cells. rEnSAG -CAP afforded an immune protection against E. necatrix. The ACI value was 164.99 in the chickens immunized with 200 µg rEnSAG -CAP . Chickens immunized with rEnSAG -CAP had a significantly higher antigen-specific serum IgY response (P < 0.0001). The data indicates that EnSAG -CAP could serve as a potential candidate antigen for the development of a recombinant coccidiosis vaccine., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Eimeria spp. induce host interleukin (IL)-4 production, a potent immune regulator, during coccidiosis to evade immune responses. Dietary anti-IL-4 may preserve bird performance during challenge; however, specific mechanisms have not been investigated. Study objectives were to develop peptide-specific anti-IL-4 antibodies and evaluate immune cell profiles and the cecal microbiota during Eimeria challenge. Four candidate IL-4 peptides were selected based on antigenicity and location. Hens were injected with conjugated peptide or carrier-only control (3/injection), eggs were collected post-vaccination and yolks were pooled by peptide before freeze-drying. On d 0, 300 Ross 708 broilers were placed in floor pens (10/pen) and assigned to 5 diets consisting of basal diet + 2% egg yolk powder containing antibodies against 1 of 4 target peptides or carrier-only control for 14-d starter and grower periods (28 d total). Baseline blood and cecal contents were collected on d 14 (6 birds/diet) before half the remainder were inoculated with 10X Coccivac-B52 (Merck Animal Health, Kenilworth, NJ). Body weight (BW) and feed intake (FI) were recorded weekly and blood and cecal samples were collected at 3, 7, and 14 d post-inoculation (pi; 3/treatment). Immune cell profiles in peripheral blood mononuclear cells (PBMC) were evaluated flow cytometrically and cecal microbial communities determined by 16S/18S rRNA gene amplicon sequencing. Data were log-transformed when necessary and analyzed with diet, Eimeria, and timepoint fixed effects plus associated interactions (SAS 9.4; P ≤ 0.05). Anti-IL-4 did not alter baseline performance but generally increased PBMC Bu-1 + B cells 38.0 to 55.4% (P < 0.0001). Eimeria challenge reduced FI and BWG 16.1 and 30.3%, respectively, regardless of diet (P < 0.0001) with only birds fed peptide 4 antibodies not recovering feed conversion by d 28. Minimal diet-associated cecal microbiota changes were observed, indicating that anti-IL-4 effects were likely host-specific. Eimeria-challenged birds fed peptide 3 antibodies displayed minimal immune cell fluctuations compared to unchallenged counterparts, suggesting these antibodies potentially modulated intestinal immune responses to minimize systemic requirements, making them good candidates for further research., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In floor-raised broilers, coccidiosis is responsible for reducing the use of nutrients, mainly by impairing intestinal tissue function and activating the immune system. Understanding and quantifying how balanced dietary protein (BP) is used when birds are challenged will allow nutritionists to make decisions regarding challenged flocks. This study aimed to determine the effects of Eimeria maxima on broiler performance and body composition, and to calculate changes in the maintenance and efficiency of protein utilisation (Ep). A total of 2 400 male 14-day-old Cobb500 broiler chickens were randomly allotted to ten groups with six replications of 40 birds each, with a 5 × 2 factorial arrangement of treatments. Five levels of BP in reference to digestible lysine (3.6, 7.2, 10.8, 14.4, and 18.0 g/kg) were fed to unchallenged (NCH) and challenged (CH) broilers with 7 × 10 3 E. maxima sporulated oocysts from 14 to 28 days of age. Performance and body deposition were measured using a comparative slaughter technique to compare BP maintenance requirements and Ep. ANOVA followed by a posthoc test was performed to compare the effects of BP levels, challenge, and their interactions. A monomolecular model describing the responses of NCH and CH broilers to BP intake, maintenance, and maximum protein deposition was compared. There were significant interactions between body weight gain and digestible lysine intake among the factors studied. Infection had a negative impact on all variables analysed, proving the efficacy of the challenge. The maintenance did not differ between the CH and NCH groups. Increased levels of dietary BP did not recover the maximum protein deposition in CH broilers. Eimeria maxima significantly reduced Ep by a factor of 0.09 times on Ep compared to the control group. The Eimeria maxima challenge was responsible to modify the use of BP altering the body composition and impairing broilers performance., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Coccidiosis caused by Eimeria is one of the most severe chicken diseases and imposes huge economic losses to the poultry industry globally. Multi-Eimeria species coinfections are common with the most prevalent combination being mixtures of Eimeria acervulina and Eimeria tenella. Although detrimental impacts of either E. acervulina or E. tenella on chicken health are well recognized, no information is available regarding their coinfection effects so far. This study was designed to investigate the influence of coinfection with E. acervulina and E. tenella on broiler chickens. 144 one-day-old broiler chickens within each of trials (trial I or II) were divided into four groups, namely, control group (CG), E. acervulina infection group (EAIG), E. tenella infection group (ETIG) and dual (E. acervulina and E. tenella) infection group (DIG). Then, chickens were measured for weight loss, lesion scores, oocyst outputs, histological changes and expressions of pro-inflammatory (interleukin [IL]-6, IL-8 and IL-18), regulatory (IL-10 and IL-22) cytokines and Toll-like receptors (TLR; TLR2 and TLR4) as well as intestinal barrier (mucin 2 [MUC2] and fattey acid-bingding proteins 2 and 6 [FABP2 and FABP6])- and tight junction (TJ; zonula occluden-1 [ZO-1], occludin [OCLN], and claudins 1 and 5 [CLDN1 and CLDN5])-related proteins at 3, 5, 7, 10, 14 and 21 days post-infection, respectively. Our results consistently showed that although ETIG and DIG exhibited a higher level of weight loss and a more amount of oocyst excretion than EAIG, DIG had lighter lesions than EAIG in the early phase because of coinfection with E. tenella. A higher (P < 0.05) ratio of duodenal villous height to crypt depth was also observed in DIG than EAIG. Moreover, histological changes in the duodenum and cecum varied by single and dual Eimeria infections. Expressions of the intestinal barrier- and TJ-related genes of EAIG, ETIG and DIG were significantly (P < 0.05) upregulated but their levels exhibited differential changes among infected chickens. Similarly, the infected chickens showed significant (P < 0.05) inflammatory responses and higher (P < 0.05) expressions of TLRs in the intestines in comparison to CG. These results presented a comprehensive physiological, pathological and immunological characterization of E. acervulina and E. tenella coinfection in broiler chickens and also shed insights into pathogenesis of multi-coccidia coinfections., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

An experiment was carried out to evaluate the impact of mixed Eimeria challenge on skeletal health of Hy-Line W-36 pullets. A total of 540, 16-day-old pullets were randomly allocated into 5 treatment groups, including a nonchallenged control. A mixed Eimeria species solution containing 50,000 E. maxima, 50,000 E. tenella, and 250,000 E. acervulina oocysts per mL was prepared and challenged to 1 group as a high-dose treatment. The 2-fold serial dilution was done to prepare the medium-high (25,000 E. maxima; 25,000 E. tenella; 125,000 E. acervulina), the medium-low (12,500 E. maxima; 12,500 E. tenella; 62,500 E. acervulina), and the low (6,250 E. maxima; 6,250 E. tenella; 31,250 E. acervulina) dose treatments which were challenged to 3 corresponding groups, respectively. The mineral apposition rate (MAR) was measured from 0 to 14 d post inoculation (DPI) and 14 to 28 DPI using calcein injection. The microstructural architecture of the femur was analyzed using the Skyscan X-ray microtomography (microCT) on 6, 14, and 28 DPI. The results showed that the MAR decreased linearly with an increase in the challenged dose (P < 0.05) during 0 to 14 DPI. The results of microCT revealed that cortical and total BMD, BMC, bone volume (BV), and bone volume as a fraction of tissue volume (BV/TV) of femur decreased both linearly (P < 0.05). Conversely, the total number of pores increased linearly with an increase in challenge dosages on 6 and 14 DPI. Trabecular BMD, BV, BV/TV, trabecular number, and trabecular thickness decreased linearly with an increase in the challenge dosages (P < 0.05) on 6 DPI. Furthermore, Eimeria infection significantly increased the number of osteoclasts and osteoclastic activity (P = 0.001). The result of this study suggests that the mixed Eimeria challenge negatively impacts the quality of skeletal health in a linear or quadratic manner with an increase in the concentration of Eimeria oocysts. The negative impact on long bone development might be due to malabsorption, nutrient deficiency during the infection, along with oxidative stress/inflammation disrupting the balance of osteoblastic and osteoclastic cells and their functions., Competing Interests: DISCLOSURES All authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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 × 10 4 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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

We investigated the effects of supplementing arginine (Arg) and branched-chain amino acids (BCAA) in broilers fed reduced-protein diets and challenged with Eimeria spp. All birds were fed the same starter diet meeting Cobb 500 nutrient specifications from d 1 to 9. Four grower diets: positive control (PC) with 20.0% crude protein (CP); reduced-protein negative control (NC) with 17.5% CP; or NC supplemented with Arg or BCAA at 50% above recommendations (ARG or BCAA) were fed to the birds from d 9 to 28. Birds were allocated in a 2 × 4 factorial arrangement (4 diets, each with or without challenge), with 8 replicates per treatment. On d 14, the challenge groups were orally gavaged with mixed Eimeria spp. Intestinal permeability was higher (P < 0.05) in NC than PC, whereas the permeability of ARG and BCAA groups did not differ significantly from PC. On d 28, a significant interaction (P < 0.01) was observed in CD8 + : CD4 + ratios in cecal tonsils (CT), Eimeria challenge increased the ratios in all groups except for the ARG group. On d 21, a significant interaction was found for CD4 + CD25 + percentages in CT (P < 0.01) that Eimeria challenge increased the percentages only in PC and NC groups. On d 21 and 28, significant interactions (P < 0.01) were found for macrophage nitric oxide (NO) production. In nonchallenged birds, NO was higher in the ARG group than other groups, but in challenged birds, NO was higher in both ARG and BCAA groups. On d 21, a significant interaction was found for bile anticoccidial IgA concentrations (P < 0.05) that Eimeria challenge increased IgA only in NC and ARG groups. The results suggest that a reduced-protein diet exacerbates the impact of the Eimeria challenge on intestinal integrity, but this could be mitigated by Arg and BCAA supplementations. Arginine and BCAA supplementations in reduced-protein diets could be beneficial for broilers against Eimeria infection by enhancing the immune responses. The beneficial effects of Arg supplementation tended to be more pronounced compared to BCAA supplementation., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The objective of the trial was to evaluate three vaccination schemes against Clostridium perfringens (CP) alpha-toxoid through drinking water to determine if they can protect against clinical signs of necrotic enteritis and coccidiosis in broiler chickens. Three hundred 1-day-old Cobb 500 male chicks were used in 4 treatments with 10 repetitions. Each group received 1 of the following treatments over the course of 29 days: T1, no vaccination; T2, vaccination on Day 1; T3, vaccination on Day 7; and T4, vaccination on Days 7 and 17. The birds were vaccinated with inactivated CP toxoid type A, administered via drinking water. During the first 14 days, a high-protein diet (27%) consisting of corn, soy, and fish meal was fed. On Day 14 Eimeria acervulina (EA), Eimeria maxima (EMx), Eimeria tenella (ET), Eimeria necatrix , and Eimeria brunetti were used in a coccidial challenge. The field isolate CP type A was then inoculated on Days 18, 19, and 20. Ten birds were slaughtered by treatment to obtain serology samples for antibody titers and intestine samples for CP and Eimeria lesion score and gut integrity indicators. Productive performance was assessed using complete randomized design and compared statistically using the Tukey test, whereas intestinal integrity variables and antibodies against CP alpha toxin were assessed using a Kruskal-Wallis nonparametric method. The results revealed that the treatments had an effect on productive performance ( P < 0.05); T3 had better body weight and weight gain than T1. In terms of lesion score at Day 21, T4 had a lower lesion score by EA, EMx, and ET than T1. Cell desquamation in T2 was lower than in T4, and excess mucus (EM) in T1 was the worst in gut integrity indicators at Day 21. On the other hand, T2 had more EM than T3 and T4 at Day 25. In the measurement of antibodies, no statistical differences ( P > 0.05) were found. These findings indicate that vaccination on Day 7 (T3) outperformed double vaccination on Days 7 and 17 (T4) and single on Day 1 (T2), in terms of productive performance, gut integrity, and lesion scores; and on the last day of the experiment T3 had the best performance in immunology response.

The study was conducted to investigate the effects of 2 isoforms of methionine on growth performance and intestinal health induced by methionine (Met) deficiency and Eimeria infection in broilers. A total of 720 one-day old male chicks (Cobb500) were randomly allocated to 10 groups in a 2 × 5 factorial arrangement (6 reps/group, 12 birds/cage) with diets and Eimeria challenge as the main factors. Hundred percent DL-Met, 100% L-Met, 80% DL-Met, and 80% L-Met diets were formulated to meet approximately 100 or 80% of the total sulfur amino acid (TSAA) requirement with DL-Met or L-Met as Met supplementation sources. The 60% TSAA basal diet (60% Met) was formulated without Met supplementation. At d14, the challenge groups were gavaged with mixed Eimeria spp. Growth performance was recorded on d7, 14, 20 (6-day postinfection [DPI]), and 26 (12 DPI). The gut permeability was measured on 5 and 11 DPI. Antioxidant status and gene expression of immune cytokines and tight junction proteins were measured on 6 and 12 DPI. Data were analyzed by 1-way and 2-way ANOVA before and after the challenge, respectively. Orthogonal polynomial contrasts were used for post hoc comparison. Overall, the Eimeria challenge and 60% Met diet significantly reduced growth performance, antioxidant status, and mRNA expression of tight junction genes and immune cytokines. For other Met treatments, the L-Met groups had significantly higher BWG and lower FCR than the DL-Met group from d 1 to 20. The L-Met groups had less gut permeability than the DL-Met groups on 5 DPI. Compared to the 80% Met groups, the 100% Met groups reduced gut permeability. At 6 DPI, the 80% Met groups showed higher ZO1 expression than the 100% Met groups. The challenge groups had higher Muc2 expression and GSH/GSSG compared to the nonchallenge groups, and SOD activity was lower in the L-Met groups compared to the DL-Met groups at 6 DPI. The 100% Met groups had higher GPx activity than the 80% Met groups at 12 DPI. In conclusion, during coccidiosis, the 100% Met groups had better gut integrity and antioxidant status. Met supplementation in the form of L-Met improved growth performance in the starter phase and gut permeability in the challenge phase., Competing Interests: DISCLOSURES There is no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)