Effect of Dietary Benzoic Acid and Oregano Essential Oil as a Substitute for an Anti-Coccidial Agent on Growth Performance and Physiological and Immunological Responses in Broiler Chickens Challenged with Eimeria Species

1. Introduction

Avian coccidiosis, caused by Eimeria spp., is one of the vulnerable enteric diseases and is endemic in vast areas of the tropical and subtropical regions [1]. It reduces growth performance affecting chicken meat production and accounts for about 30% of the total expenses on medications and other pharmacological products used to control poultry diseases [2]. Out of seven recognized species, E. acervulina, E. necatrix, and E. tenella cause serious disease outbreaks, having no cross-immunity among them [1]. These intracellular protozoa damage gut tissues and facilitate the proliferation of intestinal pathogens, leading to reduced body weight (BW) gain due to decreased feed intake [3]. Merogony, gametogony, and sporogony occur during the life cycles of Eimeria species within the host and environment, respectively [4]. After shedding, the oocysts remain viable; hence, outbreaks of coccidiosis are primarily linked to intensive rearing conditions and environmental factors [5]. To control avian coccidiosis, many commercial anticoccidials, especially salinomycin, have been utilized extensively against various Eimeria species leading to the emergence of parasites resistant to anticoccidials, a great concern to environmental issues in this present scenario [6,7]. Consequently, commercial antimicrobial-free poultry feed supplements have been promoted for the safe animal food production process as an alternative strategy for managing coccidiosis by enhancing the chickens’ innate immune systems [8,9].

Dietary acidifier benzoic acid (BA) is one such compound that improves gut integrity [10] and modulates gastrointestinal function by affecting pH-sensitive gut pathogens [11]. The antioxidative nature of BA and its ability to scavenge reactive oxygen species (ROS) [12] make it a natural antimicrobial agent that can help mitigate coccidiosis [13], along with an immunomodulation effect [14]. BA has been shown to improve average daily weight gain and feed conversion ratio (FCR) due to better nutrient digestibility in Eimeriainfected birds [15]. Oregano essential oil (OEO), a hepatoprotective phytochemical, has been used as an environmentally friendly alternative to antibiotic growth promoters to improve intestinal health, nutrient utilization, and growth performance in broiler chickens [16], including under Eimeria-infected conditions [17,18], inducing innate immunity and improving FCR [19]. It was also reported that OEO improves the hemato–biochemical profile [20], induces phagocytic activity [21], effectively reduces Eimeria population in the gut of chickens [17], and elicits a better immune response than salinomycin [22].

It was postulated that the dietary use of both BA and OEO together would effectively exert anticoccidial and antimicrobial actions and promote growth performance while preserving physiological homeostatic mechanisms in broiler chickens infected with Eimeria and addressing the antimicrobial-resistant animal food production process issues. However, a research gap exists for the appraisal of BA and OEO on growth performance, coccidia loads, immunity, and gut health compared with commercial anticoccidials in different farm conditions. We hypothesized that BA and OEO could serve as alternatives to synthetic anticoccidial chemicals without compromising production performances in coccidia-challenged conditions. Consequently, the current experiment was conducted in the environment of a small-scale poultry farm to evaluate the efficacy of BA and OEO, either alone or together, on growth performances, hemato–biochemical profile, and immune responses in Eimeriainfected broiler chickens to combat the environmental challenges concerning antimicrobial residue in the food chains.

2. Material and Methods

2.1. Experimental Animals and Design

The experimental design and protocol were approved by the Institutional Biosafety Committee (No: FVAS/Micro-IBSC/04/22-23) and Institutional Animal Ethics Committee of West Bengal University of Animal & Fishery Sciences, Kolkata, India (No. 763/GO/ReS/ReRc-L/03/CCSEA/66/2023-24).

A total of 252 unsexed Cobb-500TM (Cobb-Vantress, Inc., Siloam Springs, AR, USA; www.cobb-vantress.com; accessed on 26 January 2022) 1-day-old broiler chicks with initial BW of 42.0 ± 2.17 g were randomly and equally divided into 36 pens, each pen containing seven chicks (Table 1). The pens were randomly assigned to 6 treatments with six pens (replicates) for each treatment (n = 6), viz., (i) negative control (NC); (ii) positive control, coccidia-challenged and non-treated (PC); (iii) supplemented with a commercial anti-coccidial drug, salinomycin (Coxistac®, Phibro Animal Health CorporationTM, Teaneck, NJ, USA; marketed by Zenex Animal Health India Pvt. Ltd., Ahmedabad, India) at 500 g/kg to obtain an effective dose of 60 mg salinomycin per kilogram in the finished feed [23] and coccidia-challenged (Sal); (iv) supplemented with BA (Hi-LRTM, Himedia Laboratories Pvt. Ltd., Thane, India) at 500 mg/kg of feed (BA) following the recommendation of EFSA FEEDAP Panel [24] and coccidia-challenged; (v) supplemented with OEO extracted from Origanum compactum (AllinTM Exporters, Noida, India) at 500 mg/kg of feed [22] and coccidia-challenged (OEO); and (vi) supplemented with BA at 500 mg/kg of feed and OEO at 500 mg/kg of feed and coccidia-challenged (B&O).

Table 1. Description of the experimental design.

Birds in all groups, except the NC, were challenged with the live sporulated oocysts (Livacox®Q; Biopharm, Research Institute of Biopharmacy and Veterinary Drugs, Pohoˇrí, Czech Republic; marketed by Hester Bioscience Ltd., Ahmadabad, India). Each bird received an oral dose of 0.1 mL, 10 times the recommended vaccine dose, consisting of 3000–5000 live sporulated oocysts in each attenuated line of E. acervulina, E. maxima, and E. tenella, and 1000 live sporulated oocysts of the attenuated line of E. necatrix in a 1% w/v aqueous solution of chloramine B. The dose of Livacox®Q was increased tenfold from its usual vaccine schedule to induce mild infection, following protocols established by Nawarathne et al. [10] and Lu et al. [25].

Birds in all groups were provided with the basal concentrates diet (Table 2) and received group-wise supplementation from day 1 with the gradual replacement of starter (1 to 14 days), grower (15 to 28 days), and finisher (29 to 35 days) diets under standard operating procedure to meet or exceed the minimum nutrient requirements recommended by the Cobb 500 Broiler Performance and Nutrition Supplement [26]. The supplemented ingredients were thoroughly mixed with the mash feed on a weekly basis using a feed mixture, and diets were kept in air-tight polythene bags.

Table 2. Ingredient and nutrient composition of the diets fed to broiler chickens in different periods.

Similar standard management practice was followed for all the groups rearing at the experimental farm under the West Bengal University of Animal and Fishery Sciences, Kolkata, India (at 22◦34′ N and 88◦24′ E). The farm was disinfected with potassium permanganate solution (1:1000) 15 days before the arrival of chicks and fumigated with potassium permanganate and formaldehyde solution. The feeding and watering utensils were thoroughly cleaned and sanitized. The chicks were given continuous light for the first two days of brooding, and they quickly adapted to their new schedule of 23 h of light and 1 h of darkness. Environmental temperature and relative humidity were measured using dry-bulb and wet-bulb thermometers (Omsons Glassware Germany, Haryana, India) throughout the study period. The temperature humidity index (THI) was calculated according to the method described by Tao and Xin [27]. The mean THI was 26.75, which falls within the comfort range for broilers [28]. All birds received vaccinations against infectious bursal disease at 12 days of age and Newcastle disease at 5 and 20 days of age.

2.2. Variables Studied

2.2.1. Growth and Feed Intake

Body weights were measured pen-wise with a digital weighing balance on days 7, 14, 21, 28, and 35 (at 6 a.m.). Weekly BW gain was calculated by subtracting the initial BW from the final BW for a particular week. The average daily BW gain (ADG) was calculated by dividing the BW by the age reared in days. The average feed intake was calculated by subtracting the cumulative unconsumed daily feed from the offered feed every week. The average daily feed intake (ADFI) was calculated by dividing the feed consumed by the number of days, and feed conversion ratio (FCR) was obtained by dividing feed intake by the live weight gain during the period.

2.2.2. Sampling

Hemato–biochemical, including enzymes and hormones; fecal microbes and oocysts; antibody titer; and immunological variables were studied at weekly intervals after coccidia challenge up to day 35, i.e., on day 21 (7 days post-infection, DPI, week 3), day 28 (14 DPI, week 4) and day 35 (21 DPI, week 5).

Blood samples (4 mL) were collected from wing vein (n = 4 per replicate) on 7, 14, and 21 DPI. The samples were distributed equally into (i) ethylenediamine tetra-acetic acid (EDTA) coated vacutainer vials (Hi-media Laboratories Pvt. Ltd., Thane, India) for hematological and immunological studies, with the plasma separated from the remaining blood and stored at −20 ◦C for further analysis, as well as (ii) non-EDTA-coated vacutainer vials for serum collection for the antibody titer study.

Birds were slaughtered (n = 2 per replicate) by cervical disarticulation to collect cecal contents for oocyst counting on 7, 14, and 21 DPI as well as jejunum tissue samples for the immune regulatory gene expression study on 7 and 21 DPI.

2.2.3. Hemato–Biochemical, Enzyme, and Endocrine Profiles

Hemato–biochemical, enzyme, and endocrine profiles were measured on 7, 14, and 21 DPI from four birds in each replicate (n = 4 per replicate) across six experimental groups. Hemoglobin (Hb) percentage following the cyanmethemoglobin method [29], packed cell volume (PCV) with Wintrobe Hematocrit tube method [30], total erythrocyte counts (TEC), and total leukocyte counts (TLC) using a hemocytometer [31], and differential leukocyte counts (DLC) with a standard hematological procedure using Leishman stain (Hi-media Laboratories Pvt. Ltd., Thane, India) were measured [31]. Blood indices were calculated from the obtained results using the specific formulas, viz., mean corpuscular volume (MCV, in femtoliter) = PCV% × 10/TEC number (millions/mm³), mean corpuscular Hb (MCH, in picograms) = Hb (g/dL) × 10/TEC number (millions/mm³), and mean corpuscular Hb concentration (MCHC, in g/dL) = Hb (g/dL) × 100/PCV (%).

Blood biochemical variables, viz., glucose, total protein, albumin, total cholesterol and creatinine, and enzymes, viz., alkaline phosphatase (ALP), aspartate transaminase (AST), and alanine transaminase (ALT), were analyzed from plasma using commercially available kits (Transasia, Bio-Medicals Ltd., Mumbai, India) as per manufacturer’s protocol.

Endocrines, viz., cortisol, T3, and T4, were determined using commercially available ELISA kits following the manufacturer’s protocol (DRG Diagnostics, Marburg, Germany).

2.2.4. Fecal Oocyst and Microbial Count, Phagocytic Activity, and Lymphocyte Proliferation Response

Oocyst output was measured in terms of oocysts per gram (OPG) of feces with the McMaster method [32] at weekly intervals on 7, 14, and 21 DPI. The samples were collected from two birds per replicate (n = 2 per replicate) across six experimental groups. The cloacal swabs were used to quantify Escherichia coli, Salmonella spp., and Lactobacillus spp. numbers with spread plate method [33] using species-specific media on 7, 14, and 21 DPI from four birds in each replicate across six experimental groups. Phagocytic activity of neutrophils was measured [34] using nitroblue tetrazolium (Hi-media Laboratories Pvt. Ltd., Thane, India), and lymphocyte proliferation response was determined [35] using the colorimetric MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) (Hi-media Laboratories Pvt. Ltd., Thane, India) on 7, 14, and 21 DPI in four birds per replicate for each group.

2.2.5. Preparation of Crude Somatic Antigen of Eimeria Species and Antibody Titer Measurement

Cecal contents were collected from the Eimeria-infected slaughtered birds on 7, 14, and 21 DPI from two birds per replicate (n = 2/replicates) across six experimental groups and the oocysts were separated using the standard sedimentation and centrifugal flotation technique [32]. The separated oocysts were sporulated using 2.5% potassium dichromate following the standard method [32]. After washing with phosphate buffer solution, the oocysts were disintegrated by vortexing with glass beads (0.05 mm size) and sonicated in an ultrasound homogenizer (BANDELIN electronic GmbH & Co. KG, Berlin, Germany) using proteinase K (Qiagen, Hilden, Germany). The sonicated material was centrifuged in a cold centrifuge (Hermle, Reichenbach, Germany). The supernatant was collected as a somatic antigen of Eimeria species and protein content was measured [36].

Immunoglobulin Y (IgY) antibody titer against the somatic antigen of Eimeria spp. was measured in the serum of experimentally infected birds with indirect ELISA (i-ELISA) method [37] on 7, 14, and 21 DPI in four birds per replicate for each group. Serial dilution of serum was prepared up to five times starting from 1:100 dilutions. The crude somatic antigen of Eimeria spp. (5 µg/well) was used as a coating antigen and rabbit anti-chicken IgY-HRPO (HiMedia, Thane, India) was used as the conjugated secondary antibody. The sera from the infected control groups and healthy control group were considered positive control and negative control, respectively. The absorbance of the wells was measured at 492 nm in an ELISA Reader (Thermo Electron Corporation, Thermo Fisher Scientific, Waltham, MA, USA).

2.2.6. Gene Expressions

Fresh tissue samples from the jejunum were collected on 7 and 21 DPI from one bird of each replicate and immersed in RNA-later solution (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −80 ◦C until used for determining the expression of interleukin 10 (IL-10), interferon-gamma (IFN-γ), and Toll-like receptor 4 (TLR-4) genes. Briefly, each tissue sample was homogenized separately in a Polytron homogenizer for RNA extraction. After homogenization, the sample was transferred to a 2.0 mL centrifuge tube, and total RNA was extracted using Tri reagent (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s instructions. The integrity of the total RNA was checked by performing agarose gel electrophoresis through the Gel documentation system (Bio-Rad Laboratories Inc., Hercules, CA, USA) and total RNA was quantified using a Nanodrop spectrophotometer (Eppendorf, Hamburg, Germany). Total RNA was treated with RNase-free DNAseI (Fermentas Life Sciences, Burlington, ON, Canada) to exclude genomic DNA contamination. Then, iScript Reverse Transcriptase cDNA synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used for synthesizing cDNA. Quantification of mRNA level was achieved via quantitative real-time polymerase chain reaction (qPCR) (CFX96, Bio-Rad Laboratories Inc., Hercules, CA, USA) using the primers specific to IL-10, IFN-γ, and TLR-4 genes along with the housekeeping gene (GAPDH) (Supplementary Table S1) [38]. The gene expression levels were calculated [39] relative to the gene expression in NC at 0 DPI.

2.3. Statistical Analysis

Data were analyzed using PROC MIXED procedures of SAS [40], and the model contained treatment, week/DPI, and treatment × week interactions as main effects and pen/bird as a random effect for all variables except for antibody titers using the following statistical model:

Y_ijk = µ + T_i + W_j + (T × W)_ij + a_k + e_ijk

where Y_ijkl = dependent variable, µ = overall mean, T_i = effect of treatment i, W_j = effect of week j , (T × W)_ij = interaction effect of treatment i and week j, a_k = random effect of pen or birds k, and e_ijk = overall residual error.

The model for antibody titer included treatment, DPI, dilution, treatment × DPI, and treatment × DPI × dilution as main effects. When an interaction effect was significant (p < 0.05), the ‘SLICE’ option in the SAS model was used to find the significant difference (p < 0.05) among treatments within a week/DPI or among DPI within a treatment. Subsequently, significant differences (p < 0.05) among the treatments within a week/DPI or among the weeks/DPIs within a treatment were detected using pairwise comparisons using Fisher’s protected least significant difference test. Microbial and oocyst counts were log10-transformed before statistical analysis. Gene expression data were log2-transformed before analysis and then back-transformed to present in the table. Statistical significance was set at p ≤ 0.05.

3. Results

3.1. Growth Performances

No mortality was recorded in any group. Among the various growth and feed utilization attributes, only ADG and ADFI differed (p < 0.05) across the treatments (Trt). All the attributes increased (p < 0.01) with age and were influenced (p < 0.01) by the interactions between Trt and DPI (Table 3). After five weeks, the BA and Sal groups exhibited higher BW and ADG than the control (NC) and PC groups, followed by the OEO and B&O groups. At the end of this study, the B&O and OEO groups had the lowest ADFI and FCR, followed by the BA and Sal groups. Throughout the trial (0–5 weeks), the broiler chickens supplemented with OEO, B&O, and BA showed the best growth performance and feed consumption, outperforming the NC group and showing comparable results to the Sal group.

Table 3. Growth performances of broiler chickens supplemented with different anticoccidial agents and challenged with coccidia.

3.2. Hematological Features

There was no (p > 0.05) treatment effect on Hb, PCV, TEC, MCV, MCH, MCHC, or TLC among the groups (Table 4). Except for MCHC, significant differences (p < 0.05) among various DPI were observed in all other variables (p < 0.01). The interaction between Trt and DPI had a strong impact on Hb (p < 0.01), MCHC (p < 0.01), and MCH (p < 0.05). After the seventh DPI, the Hb concentration decreased, particularly in the PC and Sal groups. The PCV increased starting from 14 DPI. At this time (14 DPI), the TEC decreased (p < 0.05), while the MCV and MCH increased (p < 0.05) for all groups. After 7 DPI, the MCH level rose in the Sal and PC groups. There was no notable difference (p > 0.05) in MCHC among the groups or DPI. At 14 DPI, there was a decrease (p < 0.05) in TLC. Except for monocyte counts (%), which dropped at 21 DPI, various leukocyte counts including the heterophil to lymphocyte (H/L) ratio did not differ (p > 0.05) among the treatment groups, DPI, or Trt × DPI interactions (Table 5).

Table 4. Hematological features of broiler chickens supplemented with different anticoccidial agents and challenged with coccidia.

Table 5. Differential leukocyte profiles of broiler chickens supplemented with different anticoccidial agents and challenged with coccidia.

3.3. Enzymes, Endocrines and Biochemical Profile

The concentrations of endocrines and liver enzyme profiles were similar (p > 0.05) among the treatment groups (Table 6). As DPI and age progressed, a decrease (p < 0.01) in ALP and an increase in T₃ levels were observed across the groups, with the cortisol concentration dropping from 14 DPI. The only significant (p = 0.035) interaction between Trt and DPI was found for T₃ levels. At 7 DPI, the Sal group showed a lower (p < 0.05) T₃ level than the other groups. Except for creatinine concentration and the albumin-to-globulin ratio, all biochemical profiles displayed significant changes (p < 0.05) among DPIs, while the treatment and Trt × DPI had no effect (p > 0.05) (Table 7). Plasma glucose concentration decreased during mid-infection; however, total protein, albumin, globulin, and cholesterol concentration increased at 14 DPI.

Table 6. Enzyme and endocrine profiles of broiler chickens supplemented with different anticoccidial agents and challenged with coccidia.

Table 7. Biochemical profiles of broiler chickens supplemented with different anticoccidial agents and challenged with coccidia.

3.4. Fecal Oocyst and Microbial Count, Phagocytic Activity and Lymphocyte Proliferation Response

No Eimeria spp. oocysts were found in the negative control group during the study. Fecal OPG counts were higher (p < 0.01) in the PC group than in all the supplemented groups and declined (p < 0.01) after 14 DPI (Table 8). All the bacterial counts were altered (p < 0.01) by the group, DPI, and Trt × DPI interactions throughout the post-infection period, except for the non-significant (p > 0.05) effect of DPI on Lactobacillus (Table 8). The overall Salmonella and E. coli counts were highest in the PC group and lowest in the B&O group, respectively, and they followed the following pattern: B&O < OEO < BA < Sal, NC < PC and B&O < OEO, BA < Sal, NC < PC, respectively, whereas the Lactobacillus count showed the opposite pattern: B&O > OEO > BA > Sal > NC > PC. As age progressed, the number of E. coli increased while the number of Salmonella declined gradually, a pattern consistent across all groups at each DPI. The phagocytic activity of heterophils remained constant (p > 0.05) across groups and Trt × DPI interactions; however, phagocytic activity increased (p < 0.01) as DPI advanced (Table 8). There was a difference (p < 0.01) in the lymphocyte proliferation response between the B&O and PC groups, with the B&O showing the highest response and the PC the lowest (B&O > PC > BA, OEO, Sal, and NC). DPI had no effect (p > 0.05) on lymphocyte proliferation response (Table 8).

Table 8. Fecal oocyst and microbial count, phagocytic activity, and lymphocyte proliferation response of broiler chickens supplemented with different anticoccidial agents and challenged with coccidia.

3.5. Serum IgY Titer

Eimeria-specific IgY levels, in terms of optical density at 492 nm, were detected up to a maximum of four times the dilution (D4; 1:800) in all the groups and were affected (p < 0.01) by treatment, dilutions (Dil), DPI, Trt × DPI interactions, and Trt × DPI × Dil interactions (Figure 1). The overall Eimeria-specific IgY response of different groups ranged from 0.053 to 0.426 OD, with the highest response observed in the B&O group, followed by the OEO, BA, PC, and Sal groups. The NC group had the lowest IgY level. At 7 DPI, the IgY titer responded more strongly, reaching 0.411 OD. Subsequently, the levels decreased, reaching a range of 0.293 to 0.303 OD at 21 DPI. On 7 DPI, the IgY titer was highest in the Eimeria-challenged, non-supplemented group, followed by supplemented groups with the following order: PC > Sal, BA > B&O > OEO > NC. On 14 DPI, the IgY titer altered in different groups with the following order: B&O > OEO > Sal > BA, PC > NC and on 21 DPI, BA > B&O, OEO, PC > Sal > NC. The titer in the BA group remained stable throughout the duration of post-infection; however, it gradually decreased in the Sal and PC groups while showing improvement in the B&O and OEO groups as the DPI progressed.

3.6. Relative Expression of Interleukin 10, Interferon Gamma and Toll-like Receptor 4 Genes in Jejunum

The relative expression of the IL-10 gene was highest (p < 0.01) in the Sal group, followed by the PC and NC groups, with lower expression observed in the B&O, OEO, and BA groups (Figure 2A). Although there was no difference in the relative expression of IL-10 between the DPIs, the Sal and PC groups exhibited higher expression levels, and the BA group showed lower expression in both DPIs. In the B&O and NC groups, the expression was higher at 21 DPI, whereas it was lower in the OEO group. The overall relative expression of the IFN-γ gene showed the highest (p < 0.01) level in the PC group, followed by B&O and OEO, then the Sal and BA groups, while the lowest was found in the NC group (Figure 2B). The relative expression of IFN-γ increased (p < 0.01) progressively in the B&O, OEO, and PC groups with the time following coccidia challenge; in contrast, it decreased in the Sal and BA groups and was unchanged in the NC group. The overall relative expression of the TLR-4 gene was highest (p < 0.01) in the PC group, followed by the Sal, B&O, and OEO groups, and was lowest in the BA and NC groups (Figure 2C). The relative expression of TLR-4 increased (p < 0.01) progressively as DPI increased in the NC, PC, and Sal groups but it decreased in the BA and OEO groups and did not change in the B&O group.

Figure 1. Optical density (at 492 nm) of the antibody titer level in diluted serum of broiler chickens supplemented with different anticoccidial agents and challenged with coccidia. NC = negative control chickens, PC = positive control chickens, coccidia-challenged and non-treated, Sal = coccidiachallenged chickens + salinomycin at 60 mg/kg of feed, BA = coccidia-challenged chickens + benzoic acid at 500 mg/kg of feed, OEO = coccidia-challenged + oregano essential oil at 500 mg/kg of feed, B&O = coccidia-challenged + benzoic acid at 500 mg/kg of feed + oregano essential oil at 500 mg/kg of feed, Trt = treatment, DPI = days post-infection. a,b,c,d,e Means with different letters for each column bar differ significantly (p < 0.05) among the treatments (Fisher’s protected least significant difference test)

Figure 2. Relative expression (fold change) of (A) interleukin 10, (B) interferon gamma, and (C) Toll-like receptor 4 genes in the jejunum of broiler chickens supplemented with different anticoccidial agents and challenged with coccidia. NC = negative control chickens, PC = positive control chickens, coccidia-challenged and non-treated, Sal = coccidia-challenged chickens + salinomycin at 60 mg/kg of feed, BA = coccidia-challenged chickens + benzoic acid at 500 mg/kg of feed, OEO = coccidia-challenged + oregano essential oil at 500 mg/kg of feed, B&O = coccidia-challenged + benzoic acid at 500 mg/kg of feed + oregano essential oil at 500 mg/kg of feed, DPI = days postinfection. a,b,c,d,e Means with different letters for each column bar differ significantly (p < 0.05) among the treatments (Fisher’s protected least significant difference test).

4. Discussion

4.1. Growth Performances

The higher BW and ADG in the Sal and BA groups might be due to better gut morphology and a reduced pathogenic gut-microbial population [23]. A favorable gut environment for nutrient utilization may stimulate appetite, thus resulting in a modest increase in ADFI and a decrease in FCR in the salinomycin-supplemented group [41] and BA-added group [42]. Dietary essential oils might enhance the gut-beneficial microbiota population, reduce the oocyst population, and improve gut histomorphology [22,43], resulting in better nutrient efficiency and ADG in the OEO and B&O groups of birds. The combined effect of organic acid (OA) and essential oils (EO) might improve growth performance due to better nutrient digestibility and gut health of Eimeria-challenged broilers [44] by stimulating endogenous digestive enzyme, bile, and mucus secretion [45]. Increased feed efficiency was also reported as a synergistic effect of OA + EO increasing Lactobacillus and decreasing pathogenic bacteria populations in birds [15]. In this current study, the low growth performance in the PC group might be due to Eimeria infection followed by other pathogenic bacteria in the gut [16,46] that impair the gut microstructures of chickens [47] leading to impaired digestion and nutrient absorption with higher ADFI and FCR [48].

4.2. Hematological Features

The lowest Hb concentration in PC groups at 7 DPI may be due to the mechanical disruption of cecal mucosal capillaries caused by the Eimeria schizogony contamination, causing hemorrhages [20]. A similar effect on Hb and MCH concentrations was found in the Sal group due to the acute phase of Eimeria infection [49]. A steady Hb concentration might occur in coccidia-challenged birds due to reduced hemorrhage caused by supplementation of OEO [20] and organic acid in the BA group [10]. The reduction of Hb levels in the non-infected group was unexpected and might have no relation to the study. The initial significant reduction in PCV and MCV at 7 DPI irrespective of group might be due to body fluid alteration during acute infectivity [49]. These values increased at the recovery phase from 14 DPI, which might be due to the age effect and occurrence of large-sized immature RBCs after hemorrhage [50]. The reduced levels of TEC in 14 DPI may be due to the post-effect of acute coccidial infection beyond 7 DPI [51], which was restored within a week at 21 DPI by a compensatory mechanism of erythropoiesis against blood loss during infection [52]. The pulsate changes in leukocyte count might be due to the initiation of immune reaction followed by reduced inflammatory response that leads to subsequent normalization in coccidiosis [53]. This study showed that all infected birds maintained hematological values within the normal physiological range [49].

The non-significant alteration of heterophil, basophil, lymphocyte and monocyte percentage, and H/L ratio in coccidia-challenged and non-challenged broiler chickens in different DPIs might be due to less stress in the developed coccidiosis in this present study [54]. The monocytes might be involved efficiently in Eimeria infection as a part of the host-defense mechanism [49] to remove necrotic cells and tissue debris invading microorganisms, a major phagocytic component [9]. The subsequent decrease of monocytes during the later stage indicated the resolution of the initial immune response [55].

4.3. Enzymes, Endocrines, and Biochemical Profiles

The non-significant alteration of AST, ALT, and ALP among the groups indicated that the supplementation of anticoccidials and coccidia challenge had no hepatotoxic effects on the chickens [54]. The activity of ALP gradually decreased with increasing DPI, which may be due to increased osteoblastic activity and mineralization to boost the skeletal growth in the growing phase of young birds [56].

The overall T₃ and T₄ levels of different groups were found to be lower than the findings of Moryani et al. [57], and the cortisol level was found to be in line with the reported ranges of Farahani and Hosseinian [58] in broiler chickens. T₃ synthesis is stimulated by healthy gut microbiota [59] that increase with age for optimum BW gain [60]. In this present study, the T₃ level had a positive correlation with E. coli population [61]. The role of salinomycin in T₃ synthesis in chickens has not been discussed due to the paucity of the literature; hence, the cause of decreased T₃ level only at 7 DPI in the Sal group remained unexplained. However, it was reported in sheep that salinomycin had no significant effect on plasma T₃ levels [62]. A constant level of thyroxine secretion in this present study might be controlled by the dogmatic secretion of a thyrotropin-releasing hormone from the hypothalamus influenced by the overlapping role of T₃ and cortisol to encompass a homeostatic mechanism [63,64]. Coccidia may exacerbate lipid peroxidation and increase oxidative stress along with elevated cortisol levels on the 7 DPI. This condition likely improves as the infection progresses, possibly due to a decrease in coccidia numbers [65]. Birds gradually adapted to the stressors from Eimeria infection and may have returned to baseline cortisol levels over time [66]. Therefore, it can be concluded that the physiological concentration of the triiodothyronine (T₃) hormone increased with age in broiler chickens, likely due to the influence of their gut microbiome and the reduction of Eimeria-infective stress, which down-regulated cortisol levels, and the commercial anticoccidial salinomycin was found to lower T₃ levels.

Infection-induced cortisol might cause an increase in glucose levels during the early stages of infection [67]. Gluconeogenesis may be inhibited by intestinal tract inflammation at the recovery phase in 14 DPI [65], followed by the mobilization of organic reserves to restore the insufficiency in hepatic glycogen caused by higher glucose after the recovery phase in broiler chickens [68]. The periodic alteration of the total protein and its components could be related to coccidiosis [1] and time-dependent alterations of protein catabolism related to growth and fattening in broiler chickens [69]. Alteration in cholesterol levels may be due to inflammation leading to the redistribution of lipids and lipoproteins [70].

4.4. Fecal Oocyst and Microbial Count, Phagocytic Activity, and Lymphocyte Proliferation Response

Eimeria oocyst shedding in the excreta is considered the chief determining factor concerning the intensity of coccidial infection and is potentially linked to ADG and FCR [71]. The higher oocyst shedding continued up to their merogony stage until 14 DPI and decreased gradually due to the intrinsic decline of merozoites [4,10]. Salinomycin causes the movement of cations across membranes with an influx of sodium and calcium, and an efflux of potassium resulting in changes in pH and metabolic processes within Eimeria [72]. The disruption of ion gradients across the cell membranes might lead to the suppression of Eimeria oocysts in feces [45]. Oregano essential oil might discompose the cellular membrane by its active compounds (thymol and carvacrol), inhibit ATPase activity, and release intracellular ATP of the Eimeria, followed by interference of the ionic exchange leading to death of the coccidia [73]. This present study supported that essential oils may also be effective in reducing the coccidia life cycle in naturally occurring environmental coccidiosis under free-range breeding systems [74]. Benzoic acid, like other organic acids, could reduce the OPG count by inhibiting oxidative phosphorylation or electron transport to the coccidia and subsequently disturbing the life cycle of Eimeria [10].

The gradual increase in E. coli count might be the consequence of E. tenella infection, which might favor the proliferation and survivability of pathogens [47] secreting IFN-γ and disrupting heterophil extracellular traps [75], and is a common occurrence of progression of bird’s age [76]. An initial higher count of Salmonella might be the cause of Eimeria infection [47] that decreased with the advancement of DPI and is associated with the higher population of beneficial commensal bacteria, such as Lactobacillus preventing colonization of Salmonella through competitive exclusion [77]. Higher E. coli and Salmonella counts were detected in the Eimeria-challenged PC chickens, which could be due to the creation of wounds in the intestinal epithelium initiated by E. tenella favoring the proliferation and survivability of gut pathogenic microbial populations [47] utilizing the cytokines, such as IL-10 and IFN-γ, and some Toll-like receptors [78]. The supplementation of OEO might reduce E. coli and Salmonella spp. [79], disrupting their cell membranes by secretion of reactive oxygen species [80]. The process is activated by cytokines like IFN-γ in chickens [81], causing lower E. coli and Salmonella counts in the OEO and B&O groups. Reduced levels of E. coli and Salmonella spp. in BA-supplemented coccidia-challenged chickens in this present study as well as an earlier study by Zhang et al. [13] might be attributed to a lowering effect of the bacterial intracellular pH by uncoupling the electron transport, leading to the interference of the cytoplasmic membrane structure and membrane proteins [81].

Benzoic acid reduces intestinal pH, which might facilitate thereduction of the Eimeria spp. [11] and favor Lactobacillus growth [81]. The blending effect of OEO and BA indicated a positive effect on gut health by declining Eimeria spp. and amplifying Lactobacillus spp. in broiler chickens [82,83]. The present findings signify that the combination of OEO and BA can reduce the harmful Gram-negative microbiota and stimulate the growth of beneficial microflora in broiler chickens [84] compared with the salinomycintreated group.

Phagocytic activity was modified with age [85]. During infections, heterophils are primed by IFN-γ, which promotes their binding with TLR-4 [86], which leads to higher phagocytic activity with the advancement of infection [87]. Higher free radical generation in response to Eimeria was reported earlier in mice [88], which may explain higher phagocytic activity at 7 DPI. The lymphocyte proliferation response involving T-cells in protective immunity against coccidia has been recognized to reduce oocyte excretion [89]. The infection with Eimeria species might activate antigen-specific T-cell-induced immune response [90], causing higher T lymphocyte proliferation in all the Eimeria-challenged groups. Both OEO and BA are potential antimicrobial agents, having effective immunomodulatory properties [17], and might induce lymphocyte proliferation when used alone or in combination [14]. Salinomycin might induce T-cell proliferation by inhibiting the expression and enzymatic activity of immunosuppressive indoleamine-2,3-dioxygenase [91]. Hence, the steady activation of heterophils in terms of phagocytic activity potentially boosted the immune system in the experimental chickens and had a stable dominating role of lymphocyte proliferation response in the B&O group, followed by their individual supplemented groups (BA and OEO), which resisted the coccidiosis satisfactorily.

4.5. Antibody Titer (Serum IgY)

A higher level of IgY in the PC group initially at 7 DPI might be due to the natural resistance to Eimeria developed by invading the host’s intestinal epithelial cells, which induced the production of specific antibodies owing to the excess occurrence of the sporozoites and merozoites to facilitate a stronger antibody titer [92]. The IgY level declined from 14 DPI probably due to the reduced replication and maturation of Eimeria resulting in a lower secretion of specific antibodies [92]. The IgY potentially overwhelmed the host’s immune defenses and gradually weakened with the adaptation of the parasite, leading to a decline in antibody titers 21 DPI [93]. BA could improve the humoral immunity against Eimeria-infected broilers by raising the Lactobacillus population in the gut [81], which modulated the host immune response and improved antibody production [94]. The immune-stimulating effect of OEO might influence humoral immunity along with the mononuclear phagocyte and cellular immune systems in chickens, leading to better IgY in the OEO and B&O groups [95]. The gradual decline of IgY levels with the advancement of infection in the salinomycin-supplemented chickens could be indicative of the development of resistance to the drug or its inability to stimulate the host immune response over time [96]. The presence of IgY in the non-infected NC group could possibly be the natural antibody produced at baseline levels as a part of the immune system to provide passive immunity transferred from maternal antibodies [93]. Hence, it can be concluded that the Eimeria-specific IgY titer in the infected chickens reduced after 7 DPI and OEO supplementation either alone or in combination with the BA could improve it compared with the salinomycin-supplemented chickens.

4.6. Immune Gene Expressions

Eimeria spp. may induce the secretion of IL-10 to ease their invasion into chicken epithelial cells and reduce the pro-inflammatory IFN-γ, and thus, the down-regulation of IL-10 has a significant role in minimizing inflammatory responses [97]. A similar effect was noticed in the OEO and B&O groups but not in the Sal and PC groups. The OEO has an anti-inflammatory effect and down-regulates IL-10 expression in the birds, acting as a possible antibiotic substitute to control coccidiosis [25]. The Sal group had a lower expression of IL-10 and a greater expression of IFN-γ compared with the PC, contradicting the findings of Lee et al. [98]. An early and modest induction of IL-10 might occur in the non-infected NC group under normal homeostatic conditions in the jejunum that did not negatively impact resistance to Eimeria-infection [99].

The expression of IFN-γ increased in the OEO and B&O groups, which, along with the anti-inflammatory effect, helped to minimize the pathogenic effect of coccidia [100]. In the BA group, higher IFN-γ expression might be due to the dietary supplementation of organic acid [101], which probably influenced the Lactobacillus population to stimulate IFN-γ production [102]. This present study demonstrated that IFN-γ is one of the cytokines involved in coccidiosis that directly inhibits the development of Eimeria spp. within the cells [97].

TLR-4 is a transmembrane protein involved in developing specific adaptive immunity in chickens induced by Eimeria infection to promote an inflammatory response [103]. Thus, TLR-4 expressions increased in the PC group in this present study. However, TLR-4 might also respond to E. coli and Salmonella infection [9]. TLR functions can be modified by dietary manipulation (e.g., OEO) to downregulate the TLR-4-mediated inflammatory pathways, causing a lowered expression of it in the OEO and B&O groups than in the Sal and PC groups [25]. This present study signifies that BA and OEO alone and in combination increased the expression of IFN-γ and reduced IL-10 and TLR-4 to protect the chickens from redundant inflammatory reactions that were not observed in salinomycin-treated birds.

5. Conclusions

The combined dietary supplementation of BA and OEO in chickens prevented coccidiosis largely by decreasing the OPG count, increasing Eimeria-specific IgY production, and improving immune responses and FCR. OEO-supplemented chickens accomplished better ADG, and in combination with BA, increased the beneficial Lactobacillus population and reduced harmful gut E. coli and Salmonella. Salinomycin lowered the T₃ level at the initial phase of Eimeria infection. Thus, the combined application of OEO and BA may substitute for an anti-coccidial agent, salinomycin, in controlling coccidiosis with antibioticfree animal food products. This may reduce the environmental burden of pathogenic antimicrobial resistance.

This article was originally published in Animals 2024, 14, 3008. https://doi.org/10.3390/ ani14203008. This is an Open Access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).