A microbial muramidase improves growth performance and reduces inflammatory cell infiltration in the intestine of broilers chickens under Eimeria and Clostridium perfringens challenge

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 (108 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 106 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.


INTRODUCTION
The dietary supplementation of muramidase (MUR) has shown to have positive impact on the growth performance of broiler chickens (Sais et al., 2020;Wang et al., 2021;Brugaletta et al., 2022;Goes et al., 2022).The MUR enzyme targets and specifically hydrolyzes peptidoglycans (PGN) that are present in the gastrointestinal tract (GIT) of animals, as part of the normal bacterial cell turnover (Vidal et al., 2005;Sytwala et al., 2015), rather than the antinutritional factors present in the feed.Since the PGN fragments in the GIT may be recognized by the intestinal immune cells and activate an inflammatory response (Kogut et al., 2018), it is possible to hypothesize that the dietary supplementation of MUR may provide an immunological advantage to the host in dealing with the stressor (PGN).The potential anti-inflammatory effect of this enzyme could, in fact, be translated into a nutrient sparing benefit (improved digestibility) as observed previously (Goodarzi Boroojeni et al., 2019;Goes et al., 2022;Perez-Calvo et al., 2023).
The lysis of PGN by MUR reduces inflammation, improves nutrient absorption, and redirects nutrients to animal growth (Amer et al., 2023), by 2 mechanisms, mainly: reduction of PGN itself, and modulation of inflammatory mediators release.This is mainly explained by the fact that the muropeptides, breakdown products of PGN, alter the inflammatory response in the 1 GIT (Traub et al., 2006).Amer et al. (2023) have demonstrated a positive impact of MUR supplementation on the immune system of broiler chickens by the increase in blood concentration of globulins, lysozyme, IL-10, and complement 3. The significant increase of IL-10 by more than 2 folds, even with the lowest supplementation dose of MUR, is of paramount importance in demonstrating the attenuating inflammatory effect of MUR as IL-10 is well-known for its immune regulatory/anti-inflammatory effect that drives a tolerance state during chronic inflammatory conditions.Cardoso Dal Pont et al. (2023) hypothesized that in chickens, following the innate response, the adaptive immune response is characterized by a reduction of macrophage stimulatory cytokines, and increase in chronic inflammation-related cytokines, such as IL-16 and IL-10.Therefore, evaluations during and after the immune stimulus are essential to show how MUR works during the acute inflammatory response promoted by pathogens.
In the poultry industry worldwide, Clostridium perfringens has caused major economic losses as it may cause necrotic enteritis (NE), and coccidiosis infection has been considered one of the important predisposing factors of NE.It has been reported that birds supplemented with MUR under an Eimeria challenge showed higher plasmatic carotenoids, as indicator of good gastrointestinal functionality, compared to nonsupplemented animals and similar to Enramycin (Goes et al., 2022); however, no immune assessment has been performed under such challenge.We hypothesized that the supplementation of MUR mitigates the negative effects of Eimeria and Clostridium perfringens in broiler by attenuating the inflammatory process.The objective of the present studies was to evaluate different doses of supplementation of MUR in broilers under Eimeria and/or Clostridium perfringens challenge on the growth performance, intestinal histopathology and immunohistochemistry, expression of immune-related genes, blood carotenoids concentration, and carcass yield.

Experiment 1
The experiment was conducted at the Center of Immune Responses in Poultry at the Federal University of Paran a.All the procedures followed the guidelines and were approved by the internal Animal Care Committee under number 081/2016.Animals and Experimental Design A total of 256 one-day old male Cobb 500 chicks were used in this trial.The birds were placed in battery cages in a completely randomized design.The experiment consisted of 5 treatment groups with 7 replicate cages per treatment and 8 birds per cage.Each cage contained sterilized litter, nipple drinkers, and automatic temperature control.The treatments were: nonchallenged control (NC), challenged control (CC), CC + supplementation of MUR at 25,000 LSU(F)/kg, CC + supplementation of MUR at 35,000 LSU(F)/kg, and CC + Enramycin at 10 ppm (positive control-PC).The MUR used in the present study is same used by Goes et al. (2022), and its activity and recovery rates are presented in Table 3.
The birds had ad libitum access to water and feed during the entire experimental period that lasted 28 d.The birds were allocated into different rooms with equal environmental conditions, such as negative pressure ventilation, light intensity, and geographical location inside the building.The NC birds were allocated in a separated room to avoid cross contamination.Feed in mash form was based on corn, soybean meal, and meat and bone meal to meet or exceed the requirements of the birds and following the specifications for each phase: Starter (0 −14 d) and grower (14−28 d).All the diets contained phytase (RONOZYME HiPhos GT) at 1,000 FYT/kg of feed and 40 ppm of CAROPHLL yellow 10%.Anticoccidials were not included in the basal diets (Table 1).Experimental Challenge At the day of placement, the challenged groups were given, by oral gavage, 15£ the recommended dose of coccidiosis vaccine (Bio-Coccivet R-Laborat orio Biovet Brazilian Laboratory, E. acervulina, E. brunetti, E. maxima, E. necatrix, E. praecox, E. tenella, and E. mitis), and at 10, 11, and 12 d of the experiment challenged birds were inoculated by oral gavage with a fresh broth culture of a field isolate Clostridium perfringens (10 8 CFU/bird; Belote et al., 2018).Necropsy and Sampling At 7, 14, 21 and 28 d of age 1 bird/replicate was euthanized for necropsy macroscopic evaluation, and samples collection.ISI Macroscopic Evaluation After euthanasia by cervical dislocation, the birds had their health status systematically evaluated through the "I See Inside" (ISI) methodology (Kraieski et al., 2017).Various aspects of the birds were macroscopically assessed, including locomotor, respiratory, intestinal (duodenum, jejunum, ileum, and cecum), and the gastrointestinal tract (liver, yolk, proventriculus, gizzard, and pancreas).These macroscopic evaluations were carried out through the scoring of parameters from 0 to 3, where 0 = no alteration; 1 = mild; 2 = moderate; and 3 = severe alteration.For each parameter, the applied score was multiplied by its respective impact factor, which ranged from 1 to 3, according to the negative impact of that alteration on the organ functionality (3 = highest impact).The products of these multiplications were summed up to generate an ISI Total Score for each animal, where a higher value indicates a poorer health status.ISI Histopathological Evaluation For the histopathological evaluation, samples of ileum and liver were collected during the necropsy and immediately fixed in formaldehyde 10% for at least 24 h.These samples were placed in a circulator and treated with different concentrations of alcohol and toluene.The blocks were then embedded into paraffin and cut in 3 mm slices which were stained with hematoxylin and eosin (H&E) and alcian blue.For histopathological analysis, a total of 20 villi per bird were evaluated in 10£ magnification (using 40£ magnification to confirm alterations) under optical microscope (Nikon Eclipse E200, Sao Paulo, Brazil).The ISI microscopic methodology (patent: INPI BR 1020150036019) was used to measure histologic alterations on the intestine (Belote et al., 2018), and a final intestinal health index was produced.
Similar to the macroscopic analysis, scores ranging from 0 to 3 (0 = no alteration; 3 = severe alteration) are assigned to a predefined parameters observed through optical microscopy in the samples of ileum and liver.The intestinal assessment involved the scoring of lamina propria and epithelial thickness, enterocytes proliferation, inflammatory cell infiltration in the epithelium and in the lamina propria, congestion, and the presence of Eimeria spp.oocysts in the intestinal mucosa.In the liver, the following items were scored: congestion, cell vacuolation, proliferation of bile ducts, lymphocyte infiltration, pericholangitis, and lymphocyte aggregate.The applied scores were multiplied by the parameters' respective impact factors ranging from 1 to 3, which accounts for the effect of the alterations on the organ functionality.The microscopic ISI Total Score was obtained by summing up the product of these multiplications, where a higher number indicates a poorer health status of the assessed tissues.Immunohistochemical Analysis For the immunohistochemical analysis, ileum and liver samples collected in each age were fixed into zinc solution for at least 72 h.All the samples were dehydrated, infiltrated, and embedded into paraffin following common histological routine.The slides were then incubated with 500 mL of primary specific antibody for macrophages, CD4+, or CD8+ T Lymphocytes, followed by an incubation with HRP conjugated antibody specific for the primary antibody for 30 to 60 min.The peroxidase activity was blocked by using DAB kit for immunocytochemistry (HRP-conjugated rabbit anti-mouse Ig, Dako North America, Carpimteria, CA).The slides were then counterstained with hematoxylin solution.The labeled cells were counted under an optical microscope (400£ magnification).Five fields per samples were evaluated.Immune-Related Gene Expression Ileum and liver samples collected on d 7, 14, 21, and 28 were stored in 1 mL tubes containing RNA later.Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) following manufacture's recommendations.Turbo-DNAse kit (Applied Biosystems, Foster City, CA) was sued to degrade any DNA present in the samples.RNA concentration was determined with NanoDrop Spectrophotometer (Thermo Scientific, Bonn, Germany) and the integrity by Experion Automated Electrophoresis System (Bio-Rad, Hercules, CA).One mg of mRNA was used to produce cDNA in a 20 mL reaction volume using iScript Reverse Transcription Supermix kit (Bio-Rad, Hercules, CA).
The final qPCR reaction was performed using the iTAq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA).The cycle conditions for all primers used were as follow: initial denaturation step of 60 s at 95°C, followed by 40 cycles of annealing and extension of 30 s at 60°C.The melting profile of each sample was analyzed after every qPCR to confirm the product specificity.The results were analyzed the delta-delta equation and using an endogenous control (GAPDH; Table 2).

Statistical Analysis
All the analyzed parameters were considered significantly different at P ≤ 0.05.Parametric data were analyzed by 1-way ANOVA and Tukey's test was used for pairwise comparison between groups (P < 0.05).Nonparametric data (ISI scores) were submitted to Wilcoxon' test and the means separated by Dunn's test.The gene expression analysis was performed by t-test with Welch's correction at 10% probability.

Experiment 2
The experiment was conducted at the research center Mercolab, Cascavel, Parana, Brazil.All the procedures followed the guidelines and were approved by the internal Animal Care Committee, and the activities followed by a certified veterinarian.Animals, Experimental Design, and Diets A total of 1,120 one-day old male Cobb 500 chicks were used in this trial.The birds were placed in floor pens with fresh litter in a completely randomized design.The experiment consisted of 4 treatment groups with 8 replicate pens per treatment and 35 birds per pen.The treatments were: Control (basal diet), basal diet plus supplementation of MUR at 25,000 LSU(F)/kg, basal diet plus supplementation of MUR at 45,000 LSU(F)/kg, and a positive control (basal diet plus Enramycin at 10 ppm during starter and grower and 5 ppm during the finisher phase).The birds had ad libitum access to water and feed during the entire experimental period that lasted 42 d.The MUR used in the present study is same used by Goes et al. (2022), and its activity and recovery rates are presented in Table 3.
The feed in mash form was produced at the feed mill located on Mercolab's research farm.The diets were formulated following commercial recommendations usually used in Brazil, based on corn and soybean meal to meet or exceed the requirements of the birds, according to each phase: Starter (0−21 d), grower (21−35 d), and finisher (35−42 d), with phytase (RONOZYME HiPhos GT) at 1,000 FYT/kg of feed.CAROPHLL yellow 10% at 40 ppm and monensin during starter and Salinomycin during the grower phase were also included in the basal feed as anticoccidials (Table 1).Experimental Challenge 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).Growth Performance and Carcass Yield The birds and feed from each were weighed on d 21, 35 and 42 to calculate BWG, FI, and FCR.Mortality and weight of the dead birds were also recorded.At 42 d, the FCR was also corrected by a common BW (2.7 kg) to obtain cFCR.At 42 d, the birds from each pen were individually weighed to evaluate flock uniformity within each pen.The production efficiency index (PEI) was calculated on d 42.The PEI was calculated with the following formula: PEI = ((BW at d 42*(100 À mortality))/(42*FCR at d 42)*100).
At 42 d, 4 birds per replicate were used for carcass yield evaluation.Before slaughter, the birds were weighed, and the carcass (without feathers, head, feet, and viscera) and breast weight used to calculate carcass and breast yield, respectively.Carotenoid Determination At 21, 35, and 42 d, 2 birds per replicate were used to collect blood to measure total carotenoid concentration by using the iCheck CARO-TENE device and using iEx CAROTENE reagent vials (BioAnalytGmbH, Teltow, Germany).The total carotenoid concentration is used as an indicator of intestinal integrity and absorption capacity (Rochell et al., 2016).

Experiment 1
Growth Performance The growth performance results from 1 to 28 d are shown in Table 4.It was observed that the challenge significantly reduced the BWG of all challenged groups, regardless of the supplementation (P = 0.004).Feed intake was also affected by the treatment groups (P = 0.009) wherein the challenged birds supplemented with Enramycin showed lower FI than the nonchallenged control birds.Lastly, it was observed that the FCR was drastically impacted by the challenge (P = 0.02), but the supplementation of MUR at the 2 doses tested, and Enramycin partially counteracted this effect, wherein MUR improved the FCR by an average of 4.5 points compared to the challenged control birds.

ISI Macroscopic Analysis
The results for the ISI macroscopic evaluation at different ages are presented in Tables 5 and 6   (P = 0.02).Lastly, at 28 d of age, the supplementation of MUR and Enramycin only numerically attenuated the general score of the birds (P = 0.03), the intestinal score (P = 0.04) and the total score (P = 0.05) without significant difference from the challenged control birds.

ISI Histology Analysis
Liver The liver histopathology results are shown in Tables 7 and 8.At 7 d of age, it was observed that the supplementation of MUR at 35,000 LSU(F)/kg of feed significantly reduced the cell vacuolation score when compared to the challenged control group or the group supplemented with Enramycin (P < 0.001).This effect, added by nonsignificant reductions in other liver parameters, has reduced the ISI Total Score of challenged birds supplemented MUR at 35,000 LSU(F)/kg of feed to the same level of the nonchallenged animals (P < 0.001).At 14 d of age, supplementation of MUR at 25,000 LSU (F)/kg of feed reduced the score of congestion compared to the nonchallenged control group (P < 0.001), Enramycin reduced the proliferation of bile ducts (P = 0.002), and 25,000 LSU(F)/kg of feed of MUR and Enramycin numerically reduced the ISI Total Score (not different from the challenged control group).At 21 d, MUR at both doses and enramycin supplementation reduced the cell vacuolation (P < 0.001) and the ISI Total Scores (P < 0.001) compared to the challenged control group.At 28 d, birds supplemented with 35,000 LSU(F)/kg of feed of MUR and Enramycin showed reduced congestion score (P < 0.001), and numerical reduction in the ISI Total Score (P = 0.008) when compared to the challenged control birds.
Ileum The ileal histopathology results are shown in Tables 9 and 10.At 7 and 14 d, there was an effect of treatments on all the scores evaluated (Table 9).Looking at the ISI Total Score, one could observe that the challenge increased the score without positive effect of the supplementation of MUR nor Enramycin.At 21 and 28 d (Table 10), effects of the treatments could be observed on all the evaluated parameters, except for congestion.At 21 d, the MUR supplementation at 35,000 LSU(F)/kg of feed for challenged birds reduced the lamina propria thickness compared to nonsupplemented challenged group (P < 0.001).Moreover, animals supplemented with 35,000 LSU(F)/kg of feed of MUR exhibited numerical decrease in the score of epithelial thickness, proliferation of enterocytes inflammatory, cell infiltration of lamina propria, and goblet cells in comparison to the challenged birds (P < 0.001; P = 0.002; P = 0.01, and P < 0.001, respectively), not significantly differing from the challenged control group.
It was observed that at 28 d, the supplementation of MUR and Enramycin reduced the presence of oocysts in the epithelium (P < 0.001), and animals supplemented with 35,000 LSU(F)/kg of feed of MUR exhibited numerical decrease of the goblet cell score (P < 0.001).Despite positive numerical improvement of 35,000 LSU(F)/kg of feed of MUR and Enramycin on the ileum ISI Total Score on both 21 and 28 d, none of the supplemented groups significantly differed from the challenged control group.

Immunohistochemistry Analysis
Liver The results presented in Figure 1 showed that the challenge significantly (P < 0.001) increased the infiltration of macrophages in the liver at all the ages.The supplementation of both doses of MUR and Enramycin significantly attenuated this effect on d 7, 14, and 21.However, the supplementation of 35,000 LSU(F)/kg of feed of MUR had a strong positive effect in reducing the macrophage infiltration in the liver to same level as the nonchallenged control birds at all the ages evaluated.Additionally, the challenge significantly increased the infiltration of CD8+ lymphocyte in the liver at all ages (P < 0.001).At 7 d, the supplementation of 25,000 LSU(F)/kg of feed of MUR reduced its infiltration to the same level as nonchallenged birds and as those     lymphocyte infiltration.Lastly, at 7 and 14 d, the supplementation 25,000 LSU(F)/kg of feed of MUR promoted the highest infiltration of CD4+ lymphocyte when compared to the other treatments (P < 0.001).At 21 d, the challenged control birds showed the highest CD4+ lymphocyte infiltration in the ileum, followed by the birds supplemented with 25,000 LSU(F)/kg of feed of MUR and Enramycin (P < 0.001).No significant difference between the treatments was observed at 28 d of age.

Expression of Immune-Related Genes
The relative gene expression of IL-8 and IL-10 in the liver and ileum of chickens is shown in Figure 3.At 7 d, it was observed that the challenge upregulated the expression of IL-8 in the liver (P < 0.05), but the supplementation did not counteract this effect.However, at d 14, the supplementation of 35,000 LSU(F)/kg of feed of MUR and Enramycin prevented the upregulation of IL-8 in the liver which was not different from the nonchallenged control group.Additionally, the challenged upregulated the expression of IL-10 in the liver at d 14, but the supplementation of MUR at both doses and Enramycin counteracted this effect.
In the ileum, it was observed that the challenge upregulated the expression of IL-8 on d 7 and 21, and the supplementation of MUR at both doses and Enramycin counteracted this effect, but only at d 21.The challenge also increased the expression of IL-10 in the ileum at the 3 d evaluated, and only the supplementation of Enramycin, at d 21, reduced its expression to the same level as the nonchallenged control group.

Experiment 2
Growth Performance, Carcass Yield, and Blood Carotenoids The growth performance results of the Experiment 2 are shown in Table 11.It was observed that the BWG of the birds supplemented with MUR at both dosages was significantly improved, especially at the 1 to 35-day and 1 to 42-day periods (P < 0.05), when compared to the challenged control or supplemented with Enramycin, respectively.Considering the entire experimental period, from 1 to 42 d, a linear positive effect (P < 0.001) of the MUR doses was observed on the BWG.No significant differences were observed for FI.However, the body weight corrected FCR from 1 to 42 d was significantly improved by 5.6% with the supplementation of MUR (P = 0.02) when compared to the nonsupplemented birds.Additionally, a linear positive effect of MUR doses was observed on the FCR from 1 to 42 d (P = 0.008).The improved growth performance of MUR supplemented birds, also resulted in improved PEI when compared to the birds supplemented with Enramycin (P = 0.01); in addition, a linear positive effect of MUR doses was observed on the PEI at d 42 (P = 0.008).Viability and uniformity of the flock were not different between the treatment groups (P > 0.05).
The carcass yield and total blood carotenoid results are shown in Table 12.It was observed that both MUR doses evaluated increased breast yield when compared to the nonsupplemented birds (P = 0.05).The total concentration of carotenoids was increased within the supplementation of 45,000 LSU(F)/kg of feed of MUR and Enramycin when compared to the nonsupplemented group (P < 0.001).It was also observed a linear positive effect of MUR doses on the blood carotenoids concentration at d 42 (P = 0.006).

DISCUSSION
In the studies presented herein we evaluated the impact of MUR supplementation in broiler chickens undergoing Eimeria and/or Clostridium perfringens challenge with focus on growth performance, histopathology of the liver and ileum, immune-related parameters, and blood carotenoids concentration.In Experiment 1, MUR supplementation had positive impact on several macro and microscopic aspects evaluated, and reduced the infiltration of inflammatory cells in both liver and ileum during all the ages evaluated and modulated the expression of immune-related genes in both organs.These results are of paramount importance to explain the anti-inflammatory effect of dietary MUR.In Experiment 2, targeting the evaluation of the growth performance of broilers, the immunoregulatory effect of MUR was translated into improved BWG (up to 95% of the breeder guidelines with MUR supplementation), FCR, breast yield, and absorption capacity, as measured by total blood concentration of carotenoids, especially at d 42 that allowed for the longest cumulative period of supplementation.
Regarding the macro and microscopic evaluations, it was observed that the severity of lesions observed herein agreed with previous published work by Belote et al. (2018) in both liver and ileum when using a similar challenge model; however, in the present study, we have also demonstrated that MUR reduced the severity of the lesions, especially in the liver.It is important to highlight that the supplementation of MUR reduced, at least partially, various macroscopic lesions evaluated, which most likely reflects the attenuated inflammatory response promoted by the enzyme, such as lower expression of IL-8, and lower inflammatory cell infiltration, which will be further discussed.The reduction in cell infiltration in the ileum and liver by MUR has not been previously reported, but it is an essential aspect to build a deeper knowledge around MUR supplementation for broilers undergoing an enteric challenge.
Several publications have shown the benefits in supplementing broiler chickens with the same MUR to improve the growth performance beyond their genetic potential (Sais et al., 2020;Pirgozliev et al., 2021;Brugaletta et al., 2022;Goes et al., 2022).Bortoluzzi et al. (2023) reported that MUR increased the BWG and improved the FCR of chickens.Additional effects observed were reduction of the intestinal viscosity and modulation in the diversity, composition, and predictive function of the ileal microbiota.These observations are probably indirect effects of the enzyme (degradation of PGN) in attenuating the inflammatory response, as shown in the present work, that may reduce goblet cells density, and select a more beneficial microbiota composition and function (Brugaletta et al., 2022;Bortoluzzi et al., 2023).In the context of the present work, coccidiosis is ranked as the most important disease-related issue in broiler production (Blake et al., 2020), and is a wellknown predisposing factor of necrotic enteritis, caused by C. perfringens (Prescott et al., 2016), due to the increased mucus production by the goblet cells following coccidiosis infection (Collier et al., 2008) as well as the leakage of plasma proteins into the intestinal lumen (Prescott et al., 2016).It has been demonstrated that T CD8+ lymphocytes, macrophages, and to a less extend CD4+ lymphocytes are the primary cells mediating the immune response against coccidia infection (Trout and Lillehoj, 1995).As the first line of immunological defense against infective agents, macrophages perform many functions, including phagocytosis and destruction of antigens, and secretion of cytokines that regulate the proliferation and maturation of other macrophages as well as lymphocytes (Qureshi, 1998).
Chicken macrophages respond and are activated by many foreign antigens, such as coccidia, that will lead to the production of essential nitric oxide metabolites (Qureshi, 1998) which plays import role in the parasite killing (Tan et al., 2014), as well as secretion of various proinflammatory mediators (He et al., 2011), that, if exacerbated, may lead to tissue damage.Macrophages can be activated by either Th1 cytokines, such as IFN-g, that will induce macrophages with more proinflammatory properties, or Th2 cytokines, such as IL-4 and IL-13, that will induce macrophages to display more antiinflammatory and tissue repair characteristics (He et al., 2011).IL-10, on the other hand, has been described as a cytokine that inactivates macrophages by suppressing proinflammatory cytokines (O'Garra and Vieira, 2007).In Experiment 1 from the present study, the challenge increased macrophage recruitment in both liver and ileum at all 4 time points evaluated, but the supplementation of MUR, especially at the highest-level (35,000 LSU(F)/kg of feed), showed consistent effect in reducing macrophage infiltration.These results may explain the reduced damaged in the intestine by MUR supplementation as observed in some of the macroscopic evaluation parameters.
On the other hand, T cells are important key factors in driving the immune response against coccidiosis in chickens.As reviewed by Kim et al. (2019) the increased recruitment of T cells to the site of the infection induces the production of several proinflammatory cytokines with anticoccidial effects.On the other hand, it has been suggested that coccidia parasites have evolved to stimulate the production of IL-10 to inhibit the production of proinflammatory ones (Kim et al., 2019).We showed that the supplementation of MUR reduced the infiltration of CD8+ lymphocytes in both liver and ileum, and upregulated the expression of IL-10, in the ileum mainly, and downregulated IL-8, a proinflammatory cytokine, in both liver and ileum.According to Livaditi et al. (2006) IL-8 is an important biomarker in evaluating the intensity and mortality caused by sepsis, which may be because in mammals IL-8 specifically activates neutrophils and generation of reactive oxygen species.Similar studies have demonstrated that MUR upregulated the expression of NOD2 receptors, and reduced CD3+ T lymphocytes in the duodenal cell wall (Wang et al., 2021), and increased the concentration blood IL-10 in chickens (Amer et al., 2023).These results show that MUR have protective roles during the onset of the infection with anti-inflammatory and regulatory properties which leads to lower recruitment of inflammatory cells to the site of infection.
In conclusion, it was demonstrated that MUR supplementation elicited an anti-inflammatory response in birds undergoing a NE challenge model.The main observations sustaining this conclusion are the reduction in the infiltration of macrophages and CD8+ lymphocytes in the liver and ileum of infected birds and, thus, downregulation of IL-8 and upregulation of IL-10.These effects in attenuating the inflammatory response may explain the improved growth performance of supplemented birds (Experiment 2), and improved absorption capacity, as shown by the higher blood concentration of total carotenoids.

Figure 1 .
Figure 1.Immunohistochemistry results for macrophages, CD8+, and CD4+ cell (cells/field-400£ magnification objective) in the liver of chickens at different ages according to the experimental treatment groups.Experiment 1. NC: nonchallenged control; CC: Challenged control; MUR25: Muramidase at 25,000 LSU(F)/kg of feed; MUR35: Muramidase at 35,000 LSU(F)/kg of feed; ENR: Enramycin.abc Mean with different letters in the bars within the same age are significantly different at P < 0.05 Tukey's test.

Figure 2 .
Figure 2. Immunohistochemistry results for macrophages, CD8+, and CD4+ cell (cells/field-400£ magnification objective) in the ileum of chickens at different ages according to the experimental treatment groups.Experiment 1. NC: nonchallenged control; CC: Challenged control; MUR25: Muramidase at 25,000 LSU(F)/kg of feed; MUR35: Muramidase at 35,000 LSU(F)/kg of feed; ENR: Enramycin.abc Mean with different letters in the bars within the same age are significantly different at P < 0.05 Tukey's test.

Figure 3 .
Figure 3. Relative mRNA expression of IL-8 and IL-10 in the liver and ileum of chickens at different ages according to the experimental treatment groups.Experiment 1. NC: nonchallenged control; CC: challenged control; MUR25: muramidase at 25,000 LSU(F)/kg of feed; MUR35: muramidase at 35,000 LSU(F)/kg of feed; ENR: enramycin.** P < 0.05 in the unpaired t test with Welch's correction when compared with the NC group; *P < 0.10 in the unpaired t test with Welch's correction when compared with the NC group.

Table 1 .
Composition and calculated analysis of the experimental feeds.Experiments 1 and 2.

Table 3 .
Muramidase activity (LSU (F)/kg) and recovery (%) from the feed in starter, grower, and finisher phases in Experiments 1 and 2.

Table 2 .
Forward and reversed primer sequences used for qPCR analysis.Experiment 1.
and total carotenoid concentration were analyzed by ANOVA (P < 0.05), and the means separated by Tukey's test.Additionally, in Experiment 2, the doses of MUR supplementation were submitted to polynomial regression analysis (linear and quadratic) to estimate the dose response by using JMP 16.0.

Table 4 .
Growth performance from 1 to 28 d of age of broiler chickens according to the experimental groups.Experiment 1. Mean with different letters in the same column are significantly different at P < 0.05 Tukey's test. ab

Table 5 .
Results for the ISI macroscopic evaluation of the whole bird conducted during necropsy at 7 and 14 d of chickens according to the experimental treatment groups.Experiment 1.

Table 6 .
Results for the ISI macroscopic evaluation of the whole bird conducted during necropsy at 21 and 28 d of chickens according to the experimental treatment groups.Experiment 1.

Table 8 .
Results for the ISI histopathological evaluation of liver of chickens at 21 and 28 d according to the experimental treatment groups.Experiment 1.

Table 7 .
Results for the ISI histopathological evaluation of liver of chickens at 7 and 14 d according to the experimental treatment groups.Experiment 1.

Table 10 .
Results for the ISI histopathological evaluation of ileum of chickens at 21 and 28 d according to the experimental treatment groups.Experiment 1.

Table 9 .
Results for the ISI histopathological evaluation of ileum of chickens at 7 and 14 d according to the experimental treatment groups.Experiment 1.
4Inflammatory cell infiltration in the epithelium.5Inflammatorycell infiltration in the lamina propria. 6Congestion. 7Presence of oocysts. 8Goblet cells. 9Nonchallenged control.10 Challenged control.11 Muramidase at 25,000 LSU(F)/kg of feed.12 Muramidase at 35,000 LSU(F)/kg of feed.13 Enramycin (SEM = standard error of mean).abcd Mean with different letters in the same column are significantly different at P < 0.05 Dunn's test.

Table 11 .
Growth performance results according to the experimental treatment groups.Experiment 2.

Table 12 .
Carcass and breast yield and total blood carotenoid concentration (mg/L) at 42 d of age.