Integrated Morphological and Molecular Characterization of Echinococcus granulosus Infection in Sheep Liver: Evidence from Macroscopic, Microscopic, PCR, sequencing, and Cytokine Gene Expression Analyses

Abstract

Background: Hydatid disease caused by Echinococcus granulosus is a major zoonotic infection affecting livestock and humans, leading to significant economic and health burdens. Accurate diagnosis and understanding of host immune responses are essential for disease control.

Aim: This study aimed to characterize hydatid cyst infection in sheep liver using integrated macroscopic, microscopic, and molecular approaches, with emphasis on cytokine gene expression profiling.

Materials and Methods: A total of 60 liver samples (30 infected and 30 non-infected) were collected from sheep at Al-Diwaniyah slaughterhouse between September 2025 and March 2026. Macroscopic and microscopic examinations were performed to identify cyst morphology and protoscolices. Molecular detection was conducted using conventional PCR targeting the ND1 gene (300 bp), followed by sequencing and phylogenetic analysis. Cytokine gene expression (TNF-α, IFN-γ, IL-10) was quantified using RT-qPCR, and statistical analysis was performed using Welch’s t-test and Chi-square test.

Results: Macroscopic examination revealed hydatid cysts in 100% of infected samples, with sizes ranging from 1–5 cm and characteristic fluid-filled structures (χ² = 60.00, P = 1.1 × 10⁻¹⁴). Microscopic analysis confirmed the presence of protoscolices with rostellar hooks exclusively in infected samples (P < 0.0001). PCR amplification successfully detected E. granulosus DNA in all infected samples, producing a specific 300 bp band, while all controls were negative (χ² = 60.00, P = 1.1 × 10⁻¹⁴). Sequencing analysis demonstrated high similarity (97.98–100%) with global isolates. RT-qPCR results showed significant upregulation of TNF-α (9.13 ± 1.50 vs 1.06 ± 0.15; P = 2.04 × 10⁻²³) and IFN-γ (2.10 ± 0.20 vs 1.02 ± 0.10; P = 4.35 × 10⁻²⁸) in infected samples, while IL-10 expression showed no significant difference (P = 0.227). In sheep infected by hydatid cysts, histopathological examination showed the presence of protoscolices, cyst wall fibrous layer formation, laminated and germinal layers, inflammatory cell infiltration, granulomatous reaction, multinucleated giant cells, necrosis of hepatocytes, and fibroblast proliferation and necrosis in cysts.

Conclusion: Hydatid cyst infection in sheep liver is associated with distinct morphological alterations, reliable molecular detection, and strong pro-inflammatory cytokine responses. The integration of macroscopic, microscopic, PCR, and RT-qPCR approaches provides a comprehensive diagnostic and pathophysiological framework for understanding E. granulosus infection.