Comprehensive evaluation of carvacrol's anticoccidial activity: Integrating in vitro, in vivo, and in silico approaches using carvacrol-treated Eimeria tenella oocysts.

Abstract

The present study evaluated the in vitro oocysticidal activity of carvacrol against Eimeria tenella, assessed its pathogenicity in experimentally infected chicks, and explored its potential anticoccidial mechanisms through in silico analysis. In vitro assays demonstrated that carvacrol exhibited substantial efficacy, with 89.03 % inhibition of unsporulated oocyst sporulation and 85.5 % destruction of sporulated oocysts at a 10 % concentration. Oxidative stress markers in untreated oocysts showed elevated levels of superoxide dismutase (SOD) and glutathione peroxidase (GPx), alongside reduced malondialdehyde (MDA) levels, indicating an oxidative stress resistance likely essential for oocyst survival. The calculated LC₅₀ and LC₉₀ values of carvacrol against sporulated oocysts were 3.97 % and 10.82 %, respectively. For the in vivo evaluation, 60 chicks were divided into three groups (n = 20 per group). Birds were orally inoculated with 1 × 10⁴ oocysts previously treated with either the LC₅₀ or LC₉₀ concentration of carvacrol, while the control group received untreated oocysts. Chicks infected with carvacrol-treated oocysts exhibited significant reductions in bloody diarrhea, intestinal lesion scores, and oocyst shedding compared to the untreated control group. Histopathological examination revealed fewer developmental stages of E. tenella and minimal damage to the intestinal glands in treated groups, corroborating the anticoccidial efficacy of carvacrol. Biochemical analysis of cecal tissue showed that the untreated control group maintained higher SOD and GPx activity and lower MDA levels than the carvacrol-treated groups, further indicating a disruption of the parasite's oxidative stress defenses. Additionally, molecular docking studies demonstrated strong binding affinities between carvacrol and key E. tenella proteins, including fructose-1,6-bisphosphate aldolase (ALD), cyclin-dependent kinase (CDK), and apical membrane antigen 1 (AMA1), suggesting a possible mechanism through which carvacrol interferes with parasite survival and replication. Overall, these findings position carvacrol as a promising natural anticoccidial compound, exerting both direct oocysticidal effects and molecular interference with essential parasitic proteins, thereby offering a viable natural alternative to conventional synthetic drugs for the control of avian coccidiosis.