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Little is known about shifts in the fecal microbiome of dairy calves preceding and following the incidence of gastrointestinal disease. The objective of this cohort study was to describe the fecal microbiome of preweaned dairy calves before, during, and after gastrointestinal disease. A total of 111 Holstein dairy calves were enrolled on 2 dairies (D1 and D2) and followed until 5 weeks old. Health assessments were performed weekly and fecal samples were collected every other week. Of the 111 calves, 12 calves from D1 and 12 calves from D2 were retrospectively defined as healthy, and 7 calves from D1 and 11 calves from D2 were defined as diarrheic. Samples from these calves were sequenced targeting the 16S rRNA gene and compared based on health status within age groups and farms: healthy (0-1 week old) vs. pre-diarrheic (0-1 week old), healthy (2-3 weeks old) vs. diarrheic (2-3 weeks old), and healthy (4-5 weeks old) vs. post-diarrheic (4-5 weeks old) calves. Healthy and diarrheic samples clustered together based on age rather than health status on both farms. Based on linear discriminant analysis, a few species were identified to be differently enriched when comparing health status within age groups and farm. Among them, Bifidobacterium sp. was differently enriched in pre-diarrheic calves at D1 (0-1 week old) whereas healthy calves of the same age group and farm showed a higher abundance of Escherichia coli . Bifidobacterium sp. was identified as a biomarker of fecal samples from healthy calves (2-3 weeks old) on D1 when compared with diarrheic calves of the same age group and farm. Feces from diarrheic calves on D2 (2-3 weeks old) were characterized by taxa from Peptostreptococcus and Anaerovibrio genera whereas fecal samples of age-matched healthy calves were characterized by Collinsella aerofaciens and Bifidobacterium longum . After resolution of uncomplicated diarrhea (4-5 weeks old), Collinsella aerofaciens was more abundant in D2 calves whereas Bacteriodes uniformis was more abundant in D1 calves. Taken together, these findings suggest that the age of the preweaned calf is the major driver of changes to fecal microbiome composition and diversity even in the face of uncomplicated gastrointestinal disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Claus-Walker, Slanzon, Elder, Hinnant, Mandella, Parrish, Trombetta and McConnel.)

Bovine respiratory disease (BRD) is a leading cause of calf morbidity and mortality, and prevalence remains high despite current management practices. Differential gene expression (DGE) provides detailed insight into individual immune responses and can illuminate enriched pathways and biomarkers that contribute to disease susceptibility and outcomes. The aims of this study were to investigate differences in peripheral leukocyte gene expression in Holstein preweaned heifer calves 1) with and without BRD, and 2) across weeks of age. Calves were enrolled for this short-term longitudinal study on two commercial dairies in Washington State. Calves were assessed every two weeks throughout the pre-weaning period using clinical respiratory scoring (CRS) and thoracic ultrasonography (TUS), and blood samples were collected. Calves were selected that were either healthy (n = 10) or had BRD diagnosed by CRS (n = 7), TUS (n = 6), or both (n = 6) in weeks 5 or 7 of life). Three consecutive time point samples were analyzed for each BRD calf consisting of PRE, ONSET, and POST samples. Nineteen genes of interest were selected based on previous gene expression studies in cattle: ALOX15, BPI, CATHL6, CXCL8, DHX58, GZMB, HPGD, IFNG, IL17D, IL1R2, ISG15, LCN2, LIF, MX1, OAS2, PGLYRP1, S100A8, SELP, and TNF. Comparisons were made between age and disease time point matched BRD and healthy calves as well as between calf weeks of age. No DGE was observed between diseased and healthy calves; however, DGE was observed between calf weeks of age regardless of disease state. Developmental differences in leukocyte gene expression, phenotype, and functionality make pre-weaned calves immunologically distinct from mature cattle, and early life shifts in calf leukocyte populations likely contribute to the age-related gene expression differences we observed. Age overshadows disease impacts to influence gene expression in young calves, and immune development progresses upon a common trajectory regardless of disease during the preweaning period., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Elder et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)