Fluorescence-based detection of ß-d-glucuronidase activity for assessment of ileal granulocyte degranulation in Eimeria-challenged broilers

Heterophil granule component β-d-glucuronidase has been used to assess degranulation activity in cell culture supernatant after bacterial challenge. Adaptation of this assay for site specific degranulation in gastrointestinal (GIT) tissue was evaluated as a potential indicator of localized inflammation in response to Eimeria maxima (EM) infection. Experiments (Exp) 1–3 compared Control (C1, 2, 3) and EM-infected groups (EM1, 2, 3) by t-test (P < 0.05), while Experiment 4 used ANOVA and Dunnett’s post hoc analysis (P < 0.05) to compare C4 to EM-low (5 × 10³ oocysts), -medium (1 × 10⁴ oocysts), and -high (1.5 × 10⁴ oocysts). Intestinal scrapings were collected into RPMI 1% penicillin/streptomycin on ice, gently homogenized, and centrifuged. Supernatants were incubated with RPMI and positive control with serum opsonized zymosan for 1 h at 42°C. Samples were tested in 3 replicates and incubated with 4-methylumbelliferyl-β-d-glucuronide for 4 h at 42°C. Liberated 4-methylumbelliferone (4-MU) was quantified fluorometrically at an excitation/emission wavelength of 360/460 nm. Body weight gain (BWG) was evaluated over the infection period and was significantly suppressed in all EM groups relative to C1–3 (P < 0.05), while only EM-high BWG was lower (P = 0.008) than C4. In Exp 1 and 2, EM1 and EM2 4-MU was significantly lower than C1 and C2 (P = 0.046 and 0.009, respectively), while no differences were observed between C3 and EM3 in Exp 3 (P = 0.351). Exp 4 showed significant decrease in EM-low (P = 0.003) and EM-high (P = 0.003) 4-MU versus C4, but not EM-medium (P = 0.357). All assay positive controls were elevated relative to samples. Suppression of BWG indicates successful EM infection and supports the conclusion of a general observable decease in degranulation in the GIT of EM-challenged broilers. In future iterations, addition of correlation analyses between 4-MU values, BWG, and EM lesion scores on a per bird basis would aid in addressing variability within treatment groups. These results may provide new insight into GIT degranulation events occurring during coccidial infection.

Key Words: degranulation, Eimeria, gastrointestinal (GIT), inflammation, broiler.

Presented at the 9th Symposium on Gut Health in Production of Food Animals, St. Louis, USA, 2021. For information on the next edition, click here.