Evaluation of the Humoral Immune Response Against Eimeria spp. in Broiler Chickens Fed Thyme Powder and Thyme Essential Oil Using ELISA

Avian coccidiosis, caused by protozoan parasites of the genus Eimeria, remains one of the most economically detrimental enteric diseases in poultry production worldwide, with global losses estimated at over ۳ billion USD annually. Increasing drug resistance and regulatory limitations on anticoccidial agents have intensified the search for phytogenic feed additives with immunomodulatory and anticoccidial potential. This controlled experimental study evaluated the effects of dietary thyme (Thymus vulgaris) powder and thyme essential oil (TEO) on the humoral immune response against Eimeria spp. in broiler chickens using enzyme-linked immunosorbent assay (ELISA). A total of ۳۲۰ one-day-old Ross ۳۰۸ broiler chicks (mean initial body weight ۴۳.۱ ± ۱.۴ g) were randomly allocated to four dietary treatments (۸۰ birds/treatment; ۴ replicates of ۲۰ birds) for a ۴۲-day trial. The treatments were: (۱) Control: basal corn-soybean diet without additives; (۲) TP۱۰: basal diet + ۱۰ g/kg thyme powder; (۳) TEO۲۰۰: basal diet + ۲۰۰ mg/kg thyme essential oil; and (۴) TP۵+TEO۱۰۰: basal diet + ۵ g/kg thyme powder + ۱۰۰ mg/kg TEO. On day ۲۱, all birds were orally challenged with a mixed suspension of sporulated E. tenella, E. acervulina, and E. maxima oocysts at a dose of ۵ × ۱۰⁴ oocysts/bird (approximately ۱.۷ × ۱۰⁴ oocysts of each species). Serum samples were collected on days ۲۱ (pre-challenge), ۲۸, ۳۵, and ۴۲. Anti-Eimeria IgG concentrations were quantified using an indirect ELISA, and antibody response was expressed as optical density at ۴۵۰ nm (OD۴۵۰) and sample-to-positive (S/P) ratio. Fecal oocyst shedding was assessed on days ۲۷-۳۱ using the McMaster counting technique and expressed as oocysts per gram (OPG). Before challenge (day ۲۱), there were no significant differences among groups in baseline antibody levels (mean OD۴۵۰ = ۰.۴۱ ± ۰.۰۵; P > ۰.۰۵), confirming comparable initial immune status. After challenge, all thyme-supplemented groups showed significantly higher anti-Eimeria IgG titers than the control at all measured time points (P < ۰.۰۵). On day ۲۸ (۷ days post-challenge), mean OD۴۵۰ values were ۱.۱۸ ± ۰.۰۸ (Control), ۱.۴۳ ± ۰.۱۰ (TP۱۰), ۱.۵۱ ± ۰.۰۹ (TEO۲۰۰), and ۱.۶۷ ± ۰.۱۲ (TP۵+TEO۱۰۰), corresponding to increases of ۲۱.۲%, ۲۸.۰%, and ۴۱.۵% over control, respectively. The peak humoral response was observed on day ۳۵ (۱۴ days post-challenge), when OD۴۵۰ values reached ۱.۴۲ ± ۰.۰۹, ۱.۷۱ ± ۰.۱۱, ۱.۸۲ ± ۰.۱۰, and ۱.۹۴ ± ۰.۱۱ for Control, TP۱۰, TEO۲۰۰, and TP۵+TEO۱۰۰, respectively. At this time point, the combined TP۵+TEO۱۰۰ treatment increased antibody response by ۳۶.۶% relative to control, while TP۱۰ and TEO۲۰۰ increased it by ۲۰.۴% and ۲۸.۲%, respectively (P < ۰.۰۰۱). By day ۴۲, antibody levels remained elevated in thyme-supplemented groups (OD۴۵۰ = ۱.۳۶ ± ۰.۰۷, ۱.۶۳ ± ۰.۰۸, ۱.۷۴ ± ۰.۰۹, and ۱.۸۸ ± ۰.۱۰ for Control, TP۱۰, TEO۲۰۰, and TP۵+TEO۱۰۰), indicating a sustained humoral response up to ۲۱ days post-challenge. Coccidial oocyst shedding showed a corresponding reduction in thyme-fed birds. Mean total OPG values (days ۲۷-۳۱) were ۱۴۲ ± ۱۲ × ۱۰³ (Control), ۱۰۱ ± ۹ × ۱۰³ (TP۱۰), ۹۲ ± ۸ × ۱۰³ (TEO۲۰۰), and ۸۴ ± ۷ × ۱۰³ (TP۵+TEO۱۰۰). Thus, thyme powder alone reduced oocyst excretion by ۲۸.۹%, thyme essential oil alone by ۳۵.۲%, and the combined treatment by ۴۰.۸% compared with control (P < ۰.۰۵). In addition, thyme supplementation tended to improve overall performance: final body weight at day ۴۲ increased from ۲۳۸۵ ± ۵۴ g in the control group to ۲۴۶۸ ± ۴۹ g, ۲۴۹۳ ± ۵۲ g, and ۲۵۲۱ ± ۴۷ g in TP۱۰, TEO۲۰۰, and TP۵+TEO۱۰۰, respectively, while feed conversion ratio decreased from ۱.۷۸ (Control) to ۱.۷۱, ۱.۶۹, and ۱.۶۶ in the respective thyme treatments. In conclusion, dietary inclusion of thyme powder (۱۰ g/kg) and thyme essential oil (۲۰۰ mg/kg), especially in combination (۵ g/kg thyme powder + ۱۰۰ mg/kg TEO), significantly enhanced humoral immune responses against Eimeria spp. in broiler chickens, as evidenced by ۲۰-۳۷% higher ELISA antibody titers and up to ۴۱% lower oocyst shedding compared with unsupplemented controls. These results indicate that thyme-based phytogenic additives can act as effective natural immunomodulators and may represent promising alternatives or adjuncts to conventional anticoccidial strategies for coccidiosis control in broiler production systems.

کلیدواژه ها:

Broiler chicken ، coccidiosis ، Eimeria ، thyme powder ، thyme essential oil ، ELISA ، humoral immunity ، phytogenic additives