Integrated molecular, immunological, oxidative stress, and histopathological evaluation of Ascaridia galli infection in chickens.

Abstract

Ascaridia galli (A. galli) is an important intestinal nematode of chickens, but the host immune, oxidative, and pathological responses associated with natural infection remain incompletely characterized. This study aimed to provide an integrated molecular, immunological, oxidative, and histopathological evaluation of naturally occurring A. galli infection in Baladi chickens in Cairo, Egypt. A total of 200 Baladi chickens from farmers' households in rural areas of Cairo Governorate were examined between January and June 2026. Gastrointestinal helminths were recovered and identified, and 40 adult nematodes were subjected to molecular confirmation by amplification and bidirectional sequencing of the internal transcribed spacer-1 (ITS-1) region. Phylogenetic analysis was performed to assess the identity of the recovered isolate. Cytokine gene expression and intestinal oxidative stress were evaluated in infected and non-infected birds. Expression of IL-6, IL-8, IL-10, TGF-β, and IFN-γ was quantified by real-time PCR in five birds per group, while intestinal malondialdehyde (MDA), nitric oxide (NO), reduced glutathione (GSH), and catalase (CAT) were assessed as oxidative and antioxidant markers. After serological and molecular screening for concurrent infections, six infected and six non-infected chickens that were free from evidence of other infections were selected for histopathological examination, lesion scoring, intestinal morphometry, and TNF-α immunohistochemistry. A. galli was detected in 90 of 200 chickens, giving an overall prevalence of 45% ((95% CI: 38.1-52.1%). The obtained ITS-1 sequence (GenBank accession PZ363554) clustered within the A. galli clade and showed 99.57-100% nucleotide identity with reference A. galli sequences. Compared with controls, infected birds showed significant upregulation of IL-6 (28.8-fold), IL-8 (30.5-fold), IL-10 (55.9-fold), TGF-β (7.8-fold), and IFN-γ (2.2-fold), with IL-10 showing the greatest increase. Intestinal MDA and NO increased 1.44- and 1.80-fold, respectively, whereas GSH and CAT decreased to 0.76- and 0.28-fold of control levels. Histopathology revealed marked villous degeneration, sloughing and shortening, glandular degeneration, fibrosis, muscular hypertrophy, vascular alterations, and inflammatory cell infiltration. TNF-α immunoreactivity was increased in infected tissues, and morphometric analysis demonstrated alterations in villus dimensions, crypt depth, and the villus-to-crypt ratio. Natural A. galli infection was therefore associated with molecularly confirmed parasitism, altered inflammatory and regulatory cytokine expression, intestinal oxidative imbalance, and pronounced tissue injury in Baladi chickens. This work provides a foundation for experimental studies designed to unravel the causal relationships among these endpoints.