**Background:** Gastrointestinal nematodes, particularly Ascaridia galli and Heterakis gallinarum, have become widespread in non-cage laying hen systems due to oral-faecal transmission. Accurate early diagnosis is critical for controlling infections and reducing production losses. While faecal egg counts (FEC) are commonly used, they have limitations including dependence on worm fecundity. A coproantigen ELISA was recently developed but its excretion pattern and repeatability over time were unknown. This study aimed to characterize the pattern and repeatability of ascarid antigen excretion through chicken faeces and compare the diagnostic performance of coproantigen ELISA with McMaster FEC, plasma IgY ELISA, and egg yolk IgY ELISA at different weeks post-infection (wpi).
**Methods:** A total of 179 laying hens (109 Lohmann Brown Plus, 70 Lohmann Dual) were used. At 24 weeks of age, 108 hens were orally infected with 500 embryonated eggs each of A. galli and H. gallinarum, while 71 hens served as uninfected controls. At wpi 0, 2, 4, 8, 12, and 16, 29-34 hens were transferred to individual cages for 2 weeks prior to necropsy. Daily faeces were collected for four consecutive days before slaughter. Faecal samples were analysed for antigen per gram (APG) using coproantigen ELISA and for eggs per gram (EPG) using the McMaster technique (detection limit: 50 EPG). Blood and egg yolk samples were collected at necropsy for ascarid-specific IgY measurement by ELISA. Worm burden was assessed by counting, sexing, and measuring all recovered worms. Statistical analyses included repeated-measures ANOVA, intra-class correlation coefficient (ICC) for repeatability, receiver operating characteristic (ROC) analysis with DeLong test, and Pearson correlations.
**Key Results:** Significant time-dependent differences in APG between infected and control hens were observed. At wpi 2 (t(164)=0.66, P=1.00) and wpi 4 (t(164)=-3.09, P=0.094), no significant differences were found, but by wpi 6, infected hens had significantly higher APG (t(164)=-6.74, P<0.001), and differences persisted through wpi 18. The overall repeatability of APG measurements was excellent (ICC=0.91; 95% CI=0.89-0.93), though ICC was low at wpi 2 (0.08; 95% CI=0-0.47) and highest at wpi 6 (0.96; 95% CI=0.94-0.98). Plasma IgY showed significant differences between groups as early as wpi 2 (t(164)=-6.35, P<0.001). The overall diagnostic accuracy (AUC) was 0.93 for coproantigen ELISA, 0.91 for FEC, 0.88 for egg yolk IgY, and 0.83 for plasma IgY. From wpi 6 to 18, coproantigen ELISA had AUC>0.95, reaching AUC=1.00 at wpi 18. Plasma IgY showed the highest early diagnostic performance at wpi 2 (AUC=0.95). FEC had 100% specificity and 82.2% sensitivity overall, while coproantigen ELISA had 100% specificity and 76.7% sensitivity. APG showed the strongest correlations with A. galli weight (r=0.71, P<0.001) and length (r=0.69, P<0.001), while EPG correlated best with total worm counts (A. galli: r=0.44, P<0.001; H. gallinarum: r=0.32, P=0.110). APG correlated positively with adult worm counts (A. galli: r=0.6, P<0.001; H. gallinarum: r=0.52, P<0.001) but negatively with larval counts (A. galli: r=-0.59, P<0.001; H. gallinarum: r=-0.44, P<0.001).
**Clinical Implications:** The coproantigen ELISA provides a highly accurate and repeatable non-invasive method for diagnosing ascarid infections in chickens, particularly from wpi 6 onwards when antigen excretion becomes reliably detectable. Plasma IgY ELISA is superior for early detection (wpi 2), while FEC remains the best indicator of total adult worm burden post-patency. No single diagnostic method is optimal across all infection stages; the study demonstrates that the choice of diagnostic tool should be tailored to the infection stage and diagnostic objective. The authors recommend complementary use of multiple diagnostic tools for more accurate diagnosis and quantification of infections in commercial poultry settings.