Metabolomic analysis reveals altered amino acid metabolism and mechanisms underlying Eimeria infection in laying hens.

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.

Anti-coccidial efficacy of Enteromorpha prolifera polysaccharide in indigenous chickens of Northwest Ethiopia.

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 × 104 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.

Effect of orally administered B. subtilis-cNK-2 on growth performance, immunity, gut health, and gut microbiome in chickens infected with Eimeria acervulina and its potential as an alternative to antibiotics.

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 × 1010 cfu/d (CNK-O), and 5) E. acervulina-infected chickens fed a basal diet mixed with B. subtilis-cNK-2 at 1 × 1010 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.

Alterations in the gut microbiota of Eimeria infected broiler chickens fed diets supplemented with varying levels of dietary calcium and phosphorus, along with 25-hydroxycholecalciferol.

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-OHD3) 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-OHD3 (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-OHD3 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-OHD3 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-OHD3 favorably modulated the cecal microbiota in broiler chickens with/without Eimeria challenge.

Dietary advanced chelate technology-based 7-mineral supplement improves growth performance and intestinal health indicators during a mixed Eimeria challenge in broiler chickens.

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.

Aflatoxin B1 as a complicit in intestinal damage caused by Eimeria ovinoidalis in lambs: Novel insights to reveal parasite-gut battle.

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.

Insights of early feeding regime supplemented with glutamine and various levels of omega-3 in broiler chickens: growth performance, muscle building, antioxidant capacity, intestinal barriers health and defense against mixed Eimeria spp infection.

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 H2O2. 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.

Impacts of a highly pathogenic ovine Eimeria ovinoidalis on the growth of Hu lambs.

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.

Transcriptomic analysis uncovers a biphasic response to precocious Eimeria acervulina infection in chicken duodenal tissue.

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.

Broiler chicken distal jejunum microbial communities are more responsive to coccidiosis or necrotic enteritis challenge than dietary anti-interleukin-10 in a model using Salmonella Typhimurium- Eimeria maxima- Clostridium perfringens coinfection.

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 × 108Salmonella 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 1×108C. 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.

Sequencing approaches to identify distal jejunum microbial community composition and function in broiler chickens fed anti-interleukin-10 during coccidiosis and necrotic enteritis challenge.

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 (1×108 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.

Dietary Spirulina effects in Eimeria-challenged broiler chickens: growth performance, nutrient digestibility, intestinal morphology, serum biomarkers, and gene expression.

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.

In recent years, nutritional strategies for managing coccidiosis in broiler chickens have focused on feed additives, amino acids, and minerals. Spirulina, a cyanobacterium, is a feed additive rich in bioactive compounds like phycocyanin, polysaccharides, B vitamins, minerals, polyunsaturated fatty acids, and carotenoids. These compounds confer antioxidant, anti-inflammatory, and immunomodulatory properties to Spirulina. While Spirulina has been extensively explored as a health supplement in humans, its dietary application in broiler chickens and pigs is now gaining attention. Additionally, its potential to combat the Eimeria-induced effects on growth performance, nutrient digestibility, and health in birds has not been adequately addressed. Hence, the aim of this study was to investigate the effect of Spirulina on growth performance, nutrient digestibility, immune response, bone mineral deposition, and serum phosphorus in Eimeria-challenged broiler chickens. Results from this study showed that the positive properties of dietary Spirulina extend beyond its use in the diet of healthy broiler chickens as it reduced some of the negative impact of an Eimeria infection in birds.

Alternative to antimicrobial growth promoters in the diets of broilers challenged with subclinical necrotic enteritis.

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 (ViligenTM) 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.

Effect of dietary inclusion of 25-hydroxyvitamin D3 and vitamin E on performance, gut health, oxidative status, and immune response in laying hens infected with coccidiosis.

Vitamin D3 (25-hydroxyvitamin D3 (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 D3 (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.

The effect of gut microbiota-derived carnosine on mucosal integrity and immunity in broiler chickens challenged with Eimeria maxima.

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-1ß 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-1ß, 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.

Effect of dietary treatments on performance, oocysts shedding and lesion scores in broiler chickens experimentally challenged with Eimeria infection.

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 ß-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.

The global prevalence and associated risk factors of Eimeria infection in domestic chickens: A systematic review and meta-analysis.

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.

Effect of arginine supplementation on the growth performance, intestinal health, and immune responses of broilers during necrotic enteritis challenge.

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 × 104Eimeria maxima sporulated oocysts on d 14 and 1 × 108 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.

Effect of 125% and 135% arginine on the growth performance, intestinal health, and immune responses of broilers during necrotic enteritis challenge.

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 × 104Eimeria maxima sporulated oocysts on d 14 and 1 × 108 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.

Phase-specific outcmes of arginine or branched-chain amino acids supplementation in low crude protein diets on performance, nutrient digestibility, and expression of tissue protein synthesis and degradation in broiler chickens infected with mixed Eimeria spp.

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.