Short communication: Comparative gene expression analysis on the enrichment of polymorphonuclear leukocytes and gastrointestinal epithelial cells in fecal RNA from nondiarrheic neonatal dairy calves.

Abstract

Increased understanding of the biology of the gastrointestinal tract (GIT) in neonatal dairy calves during their adaptation to an extrauterine environment will decrease health problems such as diarrhea while increasing feed efficiency and average daily gain in preweaned dairy calves. Within this context, a noninvasive method, based on fecal RNA, to study the GIT in neonatal dairy calves through the isolation of RNA from fecal samples for quantitative reverse-transcription PCR analysis can provide valuable information on GIT biological adaptations during the preweaning period. We aimed to evaluate the potential enrichment of RNA from immune cells or GIT epithelial cells during fecal RNA isolation. Eight neonatal Holstein calves less than 3 wk old (14.9 ± 5.5 d of age at sampling ± standard deviation) and a fecal score of 2.0 ± 0.7 (mean ± standard deviation) were used. During a single sampling, fecal and blood samples were taken simultaneously from each calf before the morning feeding. Fecal samples were immediately frozen in liquid nitrogen until RNA isolation, whereas polymorphonuclear leukocytes (PMN) were isolated from blood samples before RNA isolation. An quantitative reverse-transcription PCR analysis was performed using a single standard curve composited of equal amounts of all samples including cDNA from fecal and PMN. The genes myeloperoxidase (MPO) and L-selectin (SELL) were selected for their specific known function in PMN, whereas keratin 8 (KRT8) and aquaporin 3 (AQP3) have been associated with epithelial enterocytes. Our results showed a contrasting gene expression profile between PMN and fecal RNA; whereas greater mRNA expression of SELL was observed in PMN, a greater KRT8 expression was observed in fecal RNA. The mRNA expression of AQP3 tended to be greater in PMN than fecal RNA. Additionally, MPO was not amplified in fecal RNA. Our findings suggest that under nondiarrheic conditions RNA isolated from stool samples of neonatal dairy calves will have a considerable number of GIT epithelial cells, which confirms the reliability of this method under these conditions. However, further research needs to be done to determine if the same effects are observed during diarrhea or throughout the preweaning period of dairy calves.