A Randomized Clinical Trial Evaluating Metabolism of Colostral and Plasma Derived Immunoglobulin G in Jersey Bull Calves

Pipkin, K.M.; Hagey, J.V.; Rayburn, M.C.; Chigerwe, M.

Methods for fecal IgG extraction were adapted from a previous method performed on fecal samples from healthy dogs. 9 Frozen fecal samples were thawed at room temperature until malleable. To decrease enzymatic degradation of the samples, only a few samples were evaluated each time. Once pliable, 1 g of feces was weighed into a 15 mL conical tube followed by addition 10 mL of phosphate buffered saline (PBS) and 50 lL of protease inhibitor cocktail. f The protease inhibitor was included to maintain the integrity of fecal IgG for later analysis. The mixture containing feces, PBS, and protease inhibitor then was homogenized using a vortex until the fecal clumps were dissolved. Samples then were centrifuged at 2,880 9 g for 5 min at 4°C to separate larger fecal particles. Aliquots of the supernatant then were collected and 5 lL of each sample was pipetted immediately into individual RID wells. A commercial bovine ultra-low-level test kit with IgG concentration determination range18-100 mg/dL was used. g The bovine ultra-low-level RID plates contained similar ingredients as the plates used for determination of plasma, serum, and colostral IgG concentrations. Aliquots (5 lL) of the provided reference serum at 3 different concentrations (10, 50 and 100 mg/dL) were pipetted into individual SRID wells on each plate used. The plates then were incubated at room temperature (20-24°C) for 24 h. The plates were read after 24 h. Fecal sample IgG concentrations were determined by comparing the diameter of the zones of precipitation with a standard curve generated by the reference serum. The regression equation generated in this manner (R 2 = 0.97-0.99) accurately predicted the inoculum fecal IgG concentration. For the purposes of this study, fecal samples with IgG concentrations <18 mg/dL were assigned an IgG concentration of 17 mg/dL. In instances where fecal IgG concentration was >100 mg/dL, SRID plates with a serum IgG determination range of 196-2,748 mg/dL were used.