Characterization of the preweaned Holstein calf fecal microbiota prior to, during, and following resolution of uncomplicated gastrointestinal disease.

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.

Differential gene expression in peripheral leukocytes of pre-weaned Holstein heifer calves with respiratory disease.

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.

Effects of a farm-specific fecal microbial transplant (FMT) product on clinical outcomes and fecal microbiome composition in preweaned dairy calves.

Gastrointestinal disease (GI) is the most common illness in pre-weaned dairy calves. Therefore, effective strategies to manipulate the microbiome of dairy calves under commercial dairy operations are of great importance to improve animal health and reduce antimicrobial usage. The objective of this study was to develop a farm-specific FMT product and to investigate its effects on clinical outcomes and fecal microbial composition of dairy calves. The FMT product was derived from feces from healthy donors (5-24 days of age) raised in the same calf ranch facility as the FMT recipients. Healthy and diarrheic calves were randomly enrolled to a control (n = 115) or FMT (n = 112) treatment group (~36 g of processed fecal matter once daily for 3 days). Fecal samples were collected at enrollment and again 9 days later after the first FMT dose. Although the FMT product was rich in organisms typically known for their beneficial probiotic properties, the FMT therapy did not prevent or ameliorate GI disease in dairy calves. In fact, calves that received FMT were less likely to recover from GI disease, and more likely to die due to GI disease complications. Fecal microbial community analysis revealed an increase in the alpha-diversity in FMT calves; however, no major differences across treatment groups were observed in the beta-diversity analysis. Calves that received FMT had higher relative abundance of an uncultured organism of the genus Lactobacillus and Lactobacillus reuteri on day 10. Moreover, FMT calves had lower relative abundance of Clostridium nexile and Bacteroides vulgatus on day 10. Our results indicate the need to have an established protocol when developing FMT products, based on rigorous inclusion and exclusion criteria for the selection of FMT donors free of potential pathogens, no history of disease or antibiotic treatment.

Fecal microbiome profiles of neonatal dairy calves with varying severities of gastrointestinal disease.

Gastrointestinal disease (GI) is the most common illness in pre-weaned dairy calves. Studies have associated the fecal microbiome composition with health status, but it remains unclear how the microbiome changes across different levels of GI disease and breeds. Our objective was to associate the clinical symptoms of GI disease with the fecal microbiome. Fecal samples were collected from calves (n = 167) of different breeds (Holstein, Jersey, Jersey-cross and beef-cross) from 4-21 d of age. Daily clinical evaluations assessed health status. Calves with loose or watery feces were diagnosed with diarrhea and classified as bright-sick (BS) or depressed-sick (DS) according to behavior. Calves with normal or semiformed feces and no clinical illness were classified as healthy (H). One hundred and three fecal samples were obtained from consistently healthy calves and 64 samples were from calves with diarrhea (n = 39 BS; n = 25 DS). The V3-V4 region of 16S rRNA gene was sequenced and analyzed. Differences were identified by a linear-mixed effects model with a negative binomial error. DS and Jersey calves had a higher relative abundance of Streptococcus gallolyticus relative to H Holstein calves. In addition, DS calves had a lower relative abundance of Bifidobacterium longum and an enrichment of Escherichia coli. Species of the genus Lactobacillus, such as an unclassified Lactobacillus, Lactobacillus reuteri, and Lactobacillus salivarius were enriched in calves with GI disease. Moreover, we created a model to predict GI disease based on the fecal microbiome composition. The presence of Eggerthella lenta, Bifidobacterium longum, and Collinsella aerofaciens were associated with a healthy clinical outcome. Although lactobacilli are often associated with beneficial probiotic properties, the presence of E. coli and Lactobacillus species had the highest coefficients positively associated with GI disease prediction. Our results indicate that there are differences in the fecal microbiome of calves associated with GI disease severity and breed specificities.

A Fixed Cohort Field Study of Gene Expression in Circulating Leukocytes From Dairy Cows With and Without Mastitis.

Specifically designed gene expression studies can be used to prioritize candidate genes and identify novel biomarkers affecting resilience against mastitis and other diseases in dairy cattle. The primary goal of this study was to assess whether specific peripheral leukocyte genes expressed differentially in a previous study of dairy cattle with postpartum disease, also would be expressed differentially in peripheral leukocytes from a diverse set of different dairy cattle with moderate to severe clinical mastitis. Four genes were selected for this study due to their differential expression in a previous transcriptomic analysis of circulating leukocytes from dairy cows with and without evidence of early postpartum disease. An additional 15 genes were included based on their cellular, immunologic, and inflammatory functions associated with resistance and tolerance to mastitis. This fixed cohort study was conducted on a conventional dairy in Washington state. Cows >50 days in milk (DIM) with mastitis (n = 12) were enrolled along with healthy cows (n = 8) selected to match the DIM and lactation numbers of mastitic cows. Blood was collected for a complete blood count (CBC), serum biochemistry, leukocyte isolation, and RNA extraction on the day of enrollment and twice more at 6 to 8-days intervals. Latent class analysis was performed to discriminate healthy vs. mastitic cows and to describe disease resolution. RNA samples were processed by the Primate Diagnostic Services Laboratory (University of Washington, Seattle, WA). Gene expression analysis was performed using the Nanostring System (Nanostring Technologies, Seattle, Washington, USA). Of the four genes (C5AR1, CATHL6, LCN2, and PGLYRP1) with evidence of upregulation in cows with mastitis, three of those genes (CATHL6, LCN2, and PGLYRP1) were investigated due to their previously identified association with postpartum disease. These genes are responsible for immunomodulatory molecules that selectively enhance or alter host innate immune defense mechanisms and modulate pathogen-induced inflammatory responses. Although further research is warranted to explain their functional mechanisms and bioactivity in cattle, our findings suggest that these conserved elements of innate immunity have the potential to bridge disease states and target tissues in diverse dairy populations.