Invited review: Impact of maternal health and nutrition on the microbiome and immune development of neonatal calves.

This comprehensive review highlights the intricate interplay between maternal factors and the co-development of the microbiome and immune system in neonatal calves. Based on human and mouse studies, multiple prenatal and postnatal factors influence this process by altering the host-associated microbiomes (gut, respiratory tract, skin), microbial colonization trajectories, and priming of the immune systems (mucosal and systemic). This review emphasizes the importance of early-life exposure, highlighting postnatal factors that work in synergy with maternal factors in further fine-tuning the co-development of the neonatal microbiome and immunity. In cattle, there is a general lack of research to identify the maternal effect on the early colonization process of neonatal calves (gut, respiratory tract) and its impact on the priming of the immune system. Past studies have primarily investigated maternal effects on the passive transfer of immunity at birth. The co-development process of the microbiome and immune system is vital for lifelong health and production in cattle. Therefore, comprehensive research beyond the traditional focus on passive immunity is an essential step in this endeavor. Calf microbiome research reports the colonization of diverse bacterial communities in newborns, which is affected by the colostrum feeding method immediately after birth. In contrast to human studies reporting a strong link between maternal and infant bacterial communities, there is a lack of evidence to clearly define cow-to-calf transmission in cattle. Maternal exposure has been shown to promote the colonization of beneficial bacteria in neonatal calves. Nonetheless, calf microbiome research lacks links to early development of the immune system. An in-depth understanding of the influence of maternal factors on microbiomes and immunity will improve the management of pregnant cows to raise immune-fit neonatal calves. It is essential to investigate the diverse effects of maternal health conditions and nutrition during pregnancy on the gut microbiome and immunity of neonatal calves through collaboration among researchers from diverse fields such as microbiology, immunology, nutrition, veterinary science, and epidemiology.

Childhood body mass index and associations with infant gut metabolites and secretory IgA: findings from a prospective cohort study.

BACKGROUND/OBJECTIVES: Differences in gut microbiota, metabolites and immune markers have been observed between individuals with and without obesity. Our study determined the temporal association between infant fecal gut metabolites, sIgA and body mass index (BMI) z score of preschool children, independent of pre/postnatal factors. SUBJECTS/METHODS: The study includes a subset of 647 infants from the CHILD Cohort Study (recruited between January 1, 2009, and December 31, 2012). Fecal metabolites and sIgA were measured at 3-4 months of age, and age and sex adjusted BMI z scores at 1 and 3 years of age. Associations between the metabolites, IgA, and child BMI z scores at age 1 and 3 years were tested using linear regression adjusted for pre/postnatal factors (breastfeeding, birthweight-for-gestational age, birthmode and IAP, solid food introduction). RESULTS: Mean BMI z score for all infants was 0.34 (SD 1.16) at 1 year (N = 647) and 0.71 (SD 1.06) at 3 years (N = 573). High fecal formate in infancy was associated with a significantly lower BMI z score (adjusted mean difference -0.23 (95% CI -0.42, -0.04)) and high butyrate was associated with a higher BMI z score (adjusted mean difference 0.21 (95% CI 0.01, 0.41)) at age 3 years only. The influence of formate and butyrate on BMI z score at age 3 were seen only in those that were not exclusively breastfed at stool sample collection (adjusted mean difference for high formate/EBF- group: -0.33 (95%CI -0.55, -0.10) and 0.25 (95% CI 0.02, 0.47) for high butyrate/EBF- group). No associations were seen between sIgA and BMI z score at age 1 or 3 years in adjusted regression models. CONCLUSION AND RELEVANCE: Differences in fecal metabolite levels in early infancy were associated with childhood BMI. This study identifies an important area of future research in understanding the pathogenesis of obesity.

Taxonomic and Functional Compositions of the Small Intestinal Microbiome in Neonatal Calves Provide a Framework for Understanding Early Life Gut Health.

A lack of information on the intestinal microbiome of neonatal calves prevents the use of microbial intervention strategies to improve calf gut health. This study profiled the taxonomic and functional composition of the small intestinal luminal microbiome of neonatal calves using whole-genome sequencing of the metagenome, aiming to understand the dynamics of microbial establishment during early life. Despite highly individualized microbial communities, we identified two distinct taxonomy-based clusters from the collective luminal microbiomes comprising a high level of either Lactobacillus or Bacteroides Among the clustered microbiomes, Lactobacillus-dominant ileal microbiomes had significantly lower abundances of Bacteroides, Prevotella, Roseburia, Ruminococcus, and Veillonella compared to the Bacteroides-dominated ileal microbiomes. In addition, the upregulated ileal genes of the Lactobacillus-dominant calves were related to leukocyte and lymphocyte chemotaxis, the cytokine/chemokine-mediated signaling pathway, and inflammatory responses, while the upregulated ileal genes of the Bacteroides-dominant calves were related to cell adhesion, response to stimulus, cell communication and regulation of mitogen-activated protein kinase cascades. The functional profiles of the luminal microbiomes also revealed two distinct clusters consisting of functions related to either high protein metabolism or sulfur metabolism. A lower abundance of Bifidobacterium and a higher abundance of sulfur-reducing bacteria (SRB) were observed in the sulfur metabolism-dominant cluster (0.2% ± 0.1%) compared to the protein metabolism-dominant cluster (12.6% ± 5.7%), suggesting an antagonistic relationship between SRB and Bifidobacterium, which both compete for cysteine. These distinct taxonomic and functional clusters may provide a framework to further analyze interactions between the intestinal microbiome and the immune function and health of neonatal calves.IMPORTANCE Dietary interventions to manipulate neonatal gut microbiota have been proposed to generate long-term impacts on hosts. Currently, our understanding of the early gut microbiome of neonatal calves is limited to 16S rRNA gene amplicon based microbial profiling, which is a barrier to developing dietary interventions to improve calf gut health. The use of a metagenome sequencing-based approach in the present study revealed high individual animal variation in taxonomic and functional abundance of intestinal microbiome and potential impacts of early microbiome on mucosal immune responses during the preweaning period. During this developmental period, age- and diet-related changes in microbial diversity, richness, density, and the abundance of taxa and functions were observed. A correlation-based approach to further explore the individual animal variation revealed potential enterotypes that can be linked to calf gut health, which may pave the way to developing strategies to manipulate the microbiome and improve calf health.

Short communication: The effect of heat treatment of bovine colostrum on the concentration of oligosaccharides in colostrum and in the intestine of neonatal male Holstein calves.

The objective of this study was to determine the effect of the heat treatment (HT, 60°C for 60 min) on the concentration of bovine colostrum oligosaccharides (bCO) in pooled bovine colostrum and the intestine of neonatal male Holstein calves after feeding. First-milking colostrum was pooled from both primiparous and multiparous cows, and half of the pooled colostrum was heat-treated at 60°C for 60 min (HC), whereas the other half was not heat-treated and remained fresh (FC). At birth, 32 male Holstein calves were randomly assigned to 1 of 3 treatment groups: (1) control calves that did not receive colostrum for the duration of the experiment and were euthanized at 6 h (NC, n = 4) or 12 h (NC, n = 4), (2) calves fed fresh colostrum (FC) and were euthanized at 6 h (FC, n = 6) or 12 h (FC, n = 6), or (3) calves fed heat-treated colostrum (HC) and euthanized at 6 h (HC, n = 6) or 12 h (HC, n = 6). All calves were fed 2 L of colostrum within 1 h after birth. At dissection, digesta of the distal jejunum, ileum, and colon was collected and analyzed by liquid chromatography-mass spectrometry to determine the concentration of bCO within each intestinal region. The heat-treated colostrum displayed numerically higher concentrations of total bCO (3,511.6 µg/g) when compared with fresh colostrum (1,329.9 µg/g), with 3'-sialyllactose being the most abundant bCO in both fresh and HT colostrum. In contrast, calves fed HT colostrum displayed a lower amount of total bCO in the distal jejunum (221.91 ± 105.3 vs. 611.26 ± 265.1 µg/g), ileum (64.97 ± 48.39 vs. 344.04 ± 216.87 µg/g), and colon (25.60 ± 13.1 vs. 267.04 ± 125.81 µg/g) at 6 h of life when compared with calves fed fresh colostrum. No differences were observed in regard to the concentrations of total bCO in the intestine of FC and HC calves at 12 h of life. It is speculated that lower concentrations of bCO in the gastrointestinal tract of HC calves at 6 h of life could be due to the early establishment of beneficial bacteria, such as Bifidobacterium, in HC calves and their subsequent metabolism of bCO as a carbon source. These findings suggest that the heat treatment of colostrum increases the amount of free bCO, which may serve as prebiotics available to microbiota within the intestine of the neonatal calf.

Understanding the gut microbiome of dairy calves: Opportunities to improve early-life gut health.

Early gut microbiota plays a vital role in the long-term health of the host. However, understanding of these microbiota is very limited in livestock species, especially in dairy calves. Neonatal calves are highly susceptible to enteric infections, one of the major causes of calf death, so approaches to improving gut health and overall calf health are needed. An increasing number of studies are exploring the microbial composition of the gut, the mucosal immune system, and early dietary interventions to improve the health of dairy calves, revealing possibilities for effectively reducing the susceptibility of calves to enteric infections while promoting growth. Still, comprehensive understanding of the effect of dietary interventions on gut microbiota-one of the key aspects of gut health-is lacking. Such knowledge may provide in-depth understanding of the mechanisms behind functional changes in response to dietary interventions. Understanding of host-microbial interactions with dietary interventions and the role of the gut microbiota during pathogenesis at the site of infection in early life is vital for designing effective tools and techniques to improve calf gut health.

Transcriptome analysis reveals regional and temporal differences in mucosal immune system development in the small intestine of neonatal calves.

BACKGROUND: Postnatal development of the mammalian mucosal immune system is crucial for responding to the rapid colonization by commensal bacteria and possible exposure to pathogens. This study analyzed expression patterns for mRNAs and their relationship with microRNAs (miRNAs) in the bovine small intestine during the critical neonatal period (0 to 42 days). This analysis revealed molecular mechanisms regulating the postnatal development of the intestinal mucosal immune system. RESULTS: Small intestine samples (jejunum and ileum) were collected from newborn male, Holstein calves immediately post-partum (n = 3) and at 7 (n = 5), 21 (n = 5), and 42 (n = 5) days of age and the transcriptomes were profiled using RNA-Seq. When analyzing all time points collectively, greater expression of genes encoding the complement functional pathway, as well as lower expression of genes encoding Toll-like receptors and NOD-like receptors were observed in the jejunum when compared to the ileum. In addition, significant changes in the expression of immune-related genes were detected within the first week post-partum in both jejunum and ileum. For example, increased expression of genes encoding tight junction proteins (claudin 1, claudin 4 and occludin), an antimicrobial peptide (Regenerating Islet-Derived 3-γ), NOD-like receptors (NACHT, LRR and PYD domain-containing protein 3), regulatory T cell marker (forkhead box P3), and both anti-inflammatory (interleukin 10) and pro-inflammatory (interleukin 8) cytokines was observed throughout the small intestine of 7-day-old calves when compared to newborn calves. Moreover, the expression of mucosal immune-related genes were either positively or negatively correlated with total bacterial population depending on both intestinal region and age. The integrated analysis of miRNAs and mRNAs supported the conclusion that miRNAs may regulate temporal changes in the expression of genes encoding tight junction proteins (miR-335), cytokines (miR-335) and bacterial recognition (miR-100) during the first week of small intestine development. CONCLUSION: The rapid development of transcriptional differences between jejunum and ileum reveal that these two intestinal regions make distinct contributions to the intestinal mucosal immune system during the early neonatal period. In addition, transcriptome analysis indicates that the first week after birth is a very dynamic developmental period for the intestinal mucosal immune system and these changes may be regulated by both miRNAs and microbial colonization. Findings from this study indicate that a detailed analysis of both the abundance and diversity of the colonizing microbiome may be necessary to understand factors regulating the rapid development of the mucosal immune system during the first week of life.

Heat-treated colostrum feeding promotes beneficial bacteria colonization in the small intestine of neonatal calves.

The present study investigated the effect of heat-treated colostrum feeding on the bacterial colonization in calf small intestine of neonatal calves within the first 12h of life. Newborn Holstein bull calves (n=32) were assigned to 3 treatment groups and fed with either fresh colostrum (FC, n=12) or heat-treated (60°C, 60 min) colostrum (HC, n=12) soon after birth, whereas the control (NC, n=8) group did not receive colostrum or water. Small intestinal tissues and contents were collected from proximal jejunum, distal jejunum, and ileum at 6 and 12h after birth, following euthanasia. Quantitative real time-PCR was used to explore the colonization of total bacteria, Lactobacillus, Bifidobacterium, and Escherichia coli. The feeding of colostrum soon after birth increased the colonization of total bacteria in calf gut within the first 12h compared with NC. In contrast, the prevalence of Lactobacillus was lower in HC and FC compared to NC. Remarkable changes in the prevalence of small intestinal tissue-attached Bifidobacterium were observed with the feeding of HC, but not that in small intestinal contents. The prevalence of Bifidobacterium was 3.2 and 5.2 fold higher in HC than FC and NC, respectively, at 6h. Although the feeding of FC did not enhance the prevalence of tissue-attached Bifidobacterium at 6h compared with NC, it displayed a gradual increase over the time that was higher than NC, but similar to that of HC at 12h. Moreover, the colonization of E. coli was drastically reduced in HC calves compared with FC and NC. Thus, the present study suggests that the feeding of HC enhances the colonization of Bifidobacterium but lessens E. coli in the calf small intestine immediately postpartum compared with that of FC and NC. The increased colonization of beneficial bacteria along with the decreased colonization of potential pathogens in calf gut may also diminish the neonatal calf diarrhea when calves are fed heat-treated colostrum soon after birth.

Taxonomic identification of commensal bacteria associated with the mucosa and digesta throughout the gastrointestinal tracts of preweaned calves.

Bacterial colonization in the gastrointestinal tracts (GIT) of preweaned calves is very important, since it can influence early development and postweaning performance and health. This study investigated the composition of the bacteria along the GIT (rumen, jejunum, ileum, cecum, and colon) of preweaned bull calves (3 weeks old) using pyrosequencing to understand the segregation of bacteria between the mucosal surface and digesta. Phylogenetic analysis revealed that a total of 83 genera belonging to 13 phyla were distributed throughout the GIT of preweaned calves, with the Firmicutes, Bacteroidetes, and Proteobacteria predominating. Quantitative PCR (qPCR) analysis of selected abundant bacterial genera (Prevotella, Bacteroides, Lactobacillus, and Faecalibacterium) revealed that their prevalence was significantly different among the GIT regions and between mucosa- and digesta-associated communities. Rumens contained the most diverse bacterial population, consisting of 47 genera, including 16 rumen-specific genera, followed by the large intestine and then the small intestine. Bacterial species richness was higher at the mucosal surface than in the local digesta, with the exception of the rumen. The majority of bacteria found on the rumen epithelial surface and within the small intestine could not be identified due to a lack of known genus-level information. Thus, future studies will be required to fully characterize the microbiome during the development of the rumens and the mucosal immune systems of newborn calves. This is the first study to analyze in depth the bacterial composition of the GIT microbiome in preweaned calves, which extends previous findings regarding early rumen colonization and bacterial segregation between mucosa- and digesta-associated microbial communities.

Characterization of the Hoof Bacterial Communities of Active Digital Dermatitis Lesions in Feedlot Cattle.

Digital dermatitis (DD) is a costly hoof infection, causing lameness and pain in feedlot cattle. DD lesions can develop nonlinearly through a series of clinical stages, which can be classified by Dopfer's M-stage scoring system. This widely adopted lesion scoring system recognizes five DD stages, where M1 (early lesion), M2 (acute ulcerative lesion), and M4.1 (chronic proliferative lesion with new developing lesion) are considered active but separate stages of the disease. This study assessed the skin surface microbiota of the active DD lesions of feedlot cattle. The DD lesions from three commercial feedlots were swabbed and then scored according to Dopfer's M-stage scoring system. Swab samples were collected from 12 M2- and 15 M4.1-stage lesions. A total of 21 control swab samples from healthy contralateral feet (DD control) were classified as stage M0. An additional six skin swabs (M0) were collected from completely healthy (CH control) cattle with no lesions. The bacterial communities of active DD lesions (M2 and M4.1) and healthy skin (M0) were profiled using 16S amplicon sequencing. Diversity analyses showed that the hoof bacterial communities of M2 and M4.1 lesions were each distinct from those of M0 skin. However, the bacterial communities between the two active lesion stages were not different from each other. A significant increase in the relative abundance of Spirochaetota and Fusobacteriota and an overall decrease in bacterial diversity contributed to the altered bacterial communities in M2 and M4.1 lesions compared to those of healthy skin (M0). Although stages M2 and M4.1 are considered clinically different stages, the lesion-associated bacterial community is similar between the two active stages.

Effect of calf starter feeding on gut microbial diversity and expression of genes involved in host immune responses and tight junctions in dairy calves during weaning transition.

Calf starters are usually offered to dairy calves to facilitate the weaning process, however, the effect of solid feed consumption on gut health has not been well studied. This study aimed to investigate the effect of calf starter feeding on the gut bacterial community and mucosal immune functions in dairy calves during weaning transition. Mucosal tissue and digesta samples were collected from rumen, jejunum, ileum, cecum, and colon upon slaughtering of calves (n=8) after feeding the experimental diets [milk replacer (MR) or milk replacer + calf starter (MR+S)] for 6 wk. Expression of toll-like receptor (TLR) 10 was downregulated along the gut, whereas TLR2 in colon and TLR6 along the gut were upregulated in MR+S-fed calves compared with MR-fed calves. Ileal TLR9 and TLR10 showed higher expression compared with the other regions regardless of the diet. Peptidoglycan recognition protein 1 demonstrated a diet- and gut-regional dependent expression pattern, whereas ß-defensin did not. The diet and gut region also affected the expression of tight junction-regulating genes claudin 4 and occludin. Bacterial diversity tended to be different between the 2 diets, whereas the bacterial density was different among gut regions and sample type. The present study revealed that changes in bacterial diversity, expression of genes encoding host mucosal immune responses, and barrier functions were associated with the MR+S diet, and suggests that solid feed consumption may alter gut microbiome and host mucosal functions during weaning transition.

Microbial Interventions to Improve Neonatal Gut Health.

The diverse pioneer microbial community colonizing the mammalian gastrointestinal tract is critical for the developing immune system. Gut microbial communities of neonates can be affected by various internal and external factors, resulting in microbial dysbiosis. Microbial dysbiosis during early life affects gut homeostasis by changing metabolic, physiological, and immunological status, which increases susceptibility to neonatal infections and long-term pathologies. Early life is crucial for the establishment of microbiota and the development of the host immune system. Therefore, it provides a window of opportunity to reverse microbial dysbiosis with a positive impact on host health. Recent attempts to use microbial interventions during early life have successfully reversed dysbiotic gut microbial communities in neonates. However, interventions with persistent effects on microbiota and host health are still limited. This review will critically discuss microbial interventions, modulatory mechanisms, their limitations, and gaps in knowledge to understand their roles in improving neonatal gut health.

Gut microbiome is linked to functions of peripheral immune cells in transition cows during excessive lipolysis.

BACKGROUND: Postpartum dairy cows experiencing excessive lipolysis are prone to severe immunosuppression. Despite the extensive understanding of the gut microbial regulation of host immunity and metabolism, its role during excessive lipolysis in cows is largely unknown. Herein, we investigated the potential links between the gut microbiome and postpartum immunosuppression in periparturient dairy cows with excessive lipolysis using single immune cell transcriptome, 16S amplicon sequencing, metagenomics, and targeted metabolomics. RESULTS: The use of single-cell RNA sequencing identified 26 clusters that were annotated to 10 different immune cell types. Enrichment of functions of these clusters revealed a downregulation of functions in immune cells isolated from a cow with excessive lipolysis compared to a cow with low/normal lipolysis. The results of metagenomic sequencing and targeted metabolome analysis together revealed that secondary bile acid (SBA) biosynthesis was significantly activated in the cows with excessive lipolysis. Moreover, the relative abundance of gut Bacteroides sp. OF04 - 15BH, Paraprevotella clara, Paraprevotella xylaniphila, and Treponema sp. JC4 was mainly associated with SBA synthesis. The use of an integrated analysis showed that the reduction of plasma glycolithocholic acid and taurolithocholic acid could contribute to the immunosuppression of monocytes (CD14+MON) during excessive lipolysis by decreasing the expression of GPBAR1. CONCLUSIONS: Our results suggest that alterations in the gut microbiota and their functions related to SBA synthesis suppressed the functions of monocytes during excessive lipolysis in transition dairy cows. Therefore, we concluded that altered microbial SBA synthesis during excessive lipolysis could lead to postpartum immunosuppression in transition cows. Video Abstract.

Saccharomyces cerevisiae boulardii accelerates intestinal microbiota maturation and is correlated with increased secretory IgA production in neonatal dairy calves.

Neonatal calves have a limited capacity to initiate immune responses due to a relatively immature adaptive immune system, which renders them susceptible to many on-farm diseases. At birth, the mucosal surfaces of the intestine are rapidly colonized by microbes in a process that promotes mucosal immunity and primes the development of the adaptive immune system. In a companion study, our group demonstrated that supplementation of a live yeast probiotic, Saccharomyces cerevisiae boulardii (SCB) CNCM I-1079, to calves from birth to 1 week of age stimulates secretory IgA (sIgA) production in the intestine. The objective of the study was to evaluate how SCB supplementation impacts the intestinal microbiota of one-week-old male calves, and how changes in the bacterial community in the intestine relate to the increase in secretory IgA. A total of 20 calves were randomly allocated to one of two treatments at birth: Control (CON, n = 10) fed at 5 g/d of carrier with no live yeast; and SCB (n = 10) fed at 5 g of live SCB per day (10 × 109 CFU/d). Our study revealed that supplementing calves with SCB from birth to 1 week of age had its most marked effects in the ileum, increasing species richness and phylogenetic diversity in addition to expediting the transition to a more interconnected bacterial community. Furthermore, LEfSe analysis revealed that there were several differentially abundant taxa between treatments and that SCB increased the relative abundance the family Eubacteriaceae, Corynebacteriaceae, Eggerthellaceae, Bacillaceae, and Ruminococcaceae. Furthermore, network analysis suggests that SCB promoted a more stable bacterial community and appears to reduce colonization with Shigella. Lastly, we observed that the probiotic-driven increase in microbial diversity was highly correlated with the enhanced secretory IgA capacity of the ileum, suggesting that the calf's gut mucosal immune system relies on the development of a stable and highly diverse microbial community to provide the necessary cues to train and promote its proper function. In summary, this data shows that supplementation of SCB promoted establishment of a diverse and interconnected microbiota, prevented colonization of Escherichia Shigella and indicates a possible role in stimulating humoral mucosal immunity.

Noncoding RNAs: Regulatory Molecules of Host-Microbiome Crosstalk.

Recent emerging evidence has revealed that regulatory noncoding RNAs (microRNAs, circular RNAs) modulate host-microbe interactions and they have been proposed as potential biomarkers of the host's response to microbiome-linked pathologies such as cancers, obesity, and neurodegenerative diseases. Interactions between microRNAs and circular RNAs, however, increase the complexity of the mechanisms that modulate host-microbe interactions. Current knowledge on these noncoding RNAs (ncRNAs) is mainly generated from well controlled germ-free or knockout (small) animal models. Application of such knowledge to effective modulation outcomes in humans (and livestock) is challenging due to the complex nature of microbiome-linked pathologies in larger outbred animals that constantly interact with the changing environment. This review critically discusses the findings of regulatory noncoding RNAs and their roles in microbiome-linked pathologies in small and large animals and provides insights on their roles as potential therapeutic agents to improve human (and livestock) health.

Effect of maternal separation and transportation stress on the bovine upper respiratory tract microbiome and the immune response to resident opportunistic pathogens.

BACKGROUND: The bovine upper respiratory tract (URT) microbiome includes opportunistic pathogens that cause respiratory disease and stress associated with maternal separation and transportation contributes to the severity of this respiratory disease. Stress is known to alter the gut microbiome but little is known regarding the effect of stress on the URT microbiota. This study used six-month old suckling beef calves to investigate whether maternal separation (weaned), by itself or combined with transportation (weaned + transport), altered the URT microbiome and host immune responses to resident opportunistic pathogens. RESULTS: Taxonomic and functional composition of the URT microbiome in suckling and weaned beef calves did not change significantly when serially sampled over a one-month period. Subtle temporal changes in the URT microbiome composition were observed in weaned + transport calves. Total bacterial density was lower (p < 0.05) on day 4 post-weaning in both the weaned and weaned + transport groups when compared to suckling calves. In addition, significant (p < 0.05) temporal changes in the density of the opportunistic pathogens, M. haemolytica and P. multocida, were observed independent of treatment but these changes did not correlate with significantly increased (p < 0.05) serum antibody responses to both of these bacteria in the weaned and weaned + transport groups. Serum antibody responses to My. bovis, another opportunistic pathogen, remained unchanged in all treatment groups. Weaning, by itself and in combination with transportation, also had significant (p < 0.05) short- (2 to 8 days post-weaning) and long-term (28 days post-weaning) effects on the expression of adrenergic receptor genes in blood leukocytes when compared to age-matched suckling beef calves. CONCLUSIONS: Maternal separation (weaning) and transportation has minor effects on the taxonomic and functional composition of the URT microbiome and temporal changes in the density of opportunistic pathogen residing in the URT did not correlate with significant changes in immune responses to these bacteria. Significant changes in adrenergic receptor expression in blood leukocytes following weaning, with or without transportation, suggests altered neuroimmune regulation should be further investigated as a mechanism by which stress can alter host-microbiome interactions for some opportunistic respiratory pathogens that reside in the URT.

A Novel Animal Model for Regional Microbial Dysbiosis of the Pioneer Microbial Community.

Pioneer microbiota colonizing the newborn gastrointestinal tract has long-lasting effects on host health. Restoration of the gut microbial community, following dysbiosis during the neonatal period, may be one strategy to prevent undesirable health outcomes linked to an altered neonatal gut microbiome. Without appropriate animal models that recreate the prolonged human neonatal developmental period it is not possible to effectively analyze interventions designed to restore regional microbial populations. Our study used a lamb model in which intestinal segments were surgically isolated (blind-ended) in fetal lambs to create early microbial dysbiosis by delaying post-natal exposure to intestinal ingesta. Intestinal segments isolated in utero retained blood flow, innervation, and lymphatic drainage through the mesenteric attachment. Continuity of the fetal gastro-intestinal tract was re-established by side-to-side anastomosis of intestine proximal and distal to each isolated intestinal segment. Microbial restoration was then implemented in neonatal lambs by reconnecting a portion of the in utero isolated intestinal segments to adjacent intestinal tract 1 and 7 days after birth. Bacterial communities colonizing the adjacent intestine, in utero isolated intestinal segments, and reconnected intestinal segments were profiled using 16S amplicon sequencing on days 1, 7, and 56 of age. The in utero isolated intestinal segments were colonized 1 day after birth but the density of active bacteria was reduced and community composition altered when compared to adjacent intestine. Proteobacteria dominated the adjacent small intestine at early time points (day 1 and day 7) with a shift to primarily Firmicutes on day 56, consistent with establishment of an anaerobic bacterial community. In contrast, Proteobacteria persisted as the predominant community for 56 days in the in utero isolated intestinal segments. There was, however, almost full restoration of the microbial community composition in the in utero isolated intestinal segments following reconnection to the adjacent intestine. The density of beneficial bacteria, especially Bifidobacterium, remained significantly lower in the reconnected intestinal segments at 56 days when compared to adjacent intestine. Post-natal persistence of a stable pioneer community (Proteobacteria) in the in utero isolated intestinal segments provides a model system to study the temporal effects of regional microbial dysbiosis throughout a prolonged neonatal period.

Regulation of rumen development in neonatal ruminants through microbial metagenomes and host transcriptomes.

BACKGROUND: In ruminants, early rumen development is vital for efficient fermentation that converts plant materials to human edible food such as milk and meat. Here, we investigate the extent and functional basis of host-microbial interactions regulating rumen development during the first 6 weeks of life. RESULTS: The use of microbial metagenomics, together with quantification of volatile fatty acids (VFAs) and qPCR, reveals the colonization of an active bacterial community in the rumen at birth. Colonization of active complex carbohydrate fermenters and archaea with methyl-coenzyme M reductase activity was also observed from the first week of life in the absence of a solid diet. Integrating microbial metagenomics and host transcriptomics reveals only 26.3% of mRNA transcripts, and 46.4% of miRNAs were responsive to VFAs, while others were ontogenic. Among these, one host gene module was positively associated with VFAs, while two other host gene modules and one miRNA module were negatively associated with VFAs. Eight host genes and five miRNAs involved in zinc ion binding-related transcriptional regulation were associated with a rumen bacterial cluster consisting of Prevotella, Bacteroides, and Ruminococcus. CONCLUSION: This three-way interaction suggests a potential role of bacteria-driven transcriptional regulation in early rumen development via miRNAs. Our results reveal a highly active early microbiome that regulates rumen development of neonatal calves at the cellular level, and miRNAs may coordinate these host-microbial interactions.

Colostrum feeding shapes the hindgut microbiota of dairy calves during the first 12 h of life.

This study evaluated the effect of feeding non-heated and heated colostrum on the mucosa- and digesta-associated microbiota in the colon of dairy calves during the first 12 h of life. Thirty-two neonatal Holstein male calves were fed: no colostrum (NC, n = 8), non-heated colostrum (FC, n = 12) and heated colostrum (HC (60 °C, 60 min), n = 12) immediately after birth. The abundances of mucosa- and digesta-associated total bacteria were higher in the colon of FC fed calves compared to those fed no colostrum (NC) at 12 h of life. Compare to NC calves, a higher proportion of mucosa- and digesta-associated Clostridium cluster XIVa and Bifidobacterium, and a lower abundance of mucosa and digesta-associated E. coli were detected in the colon of FC and HC fed calves, as well as a tentatively lower relative abundance of Escherichia-Shigella genus in colon mucosa of HC fed calves. In addition, HC calves had lower abundances of E. coli and higher abundances of Bifidobacterium in mucosa-associated microbiota than FC fed calves. Our results suggest that feeding non-heated colostrum immediately after birth benefit neonatal calves with increased Bifidobacterium and decreased opportunistic pathogenic E. coli and Escherichia-Shigella genus in the colon, and feeding heated colostrum can fortify such effects.

Fetal environment and fetal intestine are sterile during the third trimester of pregnancy.

Recent next generation sequencing studies on host-associated microbiomes generated debatable conclusions regarding the central dogma of fetal gut sterility. These observations challenge the concepts that microbial colonization of the gut begins during and after birth as well as the concept of antigen-independent prenatal maturation of mucosal-associated lymphoid tissue in ruminants and humans. The placental barrier varies markedly among mammalian species with mice and humans having haemochorial placentas (fetal tissue in direct contact with maternal blood) versus epitheliochorial placentation (maternal and fetal blood separated by six tissue layers) in ruminants. Therefore, this study re-examined the question of fetal gut sterility using the fetal lamb as a model ruminant species with the most complete placental barrier. Use of PCR and quantitative real-time PCR with three different pairs of universal bacterial primers (27 F and 1492R, HDA1 and HDA2, U2F and U2R) to amplify 16S rRNA gene did not generate detectable PCR products from samples collected from the fetal environment (placenta, amniotic fluid) and fetal intestine during the third trimester of pregnancy. Procedures to further enrich microbial DNA from total extracted DNA also resulted in no detectable genomic DNA. Moreover, use of 16S amplicon sequencing confirmed the absence of bacteria in the fetal environment during the third trimester of pregnancy. A 'No Template' control containing only PCR reagents generated sequences that could be clustered into OTUs at 97% similarity and assigned to bacterial genera, including Staphylococcus, Lactobacillus and Escherichia-Shigella. Use of multiple molecular-based approaches to profile fetal environment-associated microbiota supports the conclusion that the fetal environment and fetal intestine remain sterile during the third trimester of pregnancy. The use of appropriate controls, both positive and no template, revealed inherent contamination in reagents and that variations in the data analysis pipeline can produce artificial microbial profiles from host tissues containing low microbial biomass. Finally, these findings confirm that extensive development of gut-associated lymphoid tissue in the ruminant fetal intestine, characterized by active B cell proliferation and immunoglobulin V gene somatic mutation, is not associated with exposure to bacterial DNA and antigens.

Development and Function of the Mucosal Immune System in the Upper Respiratory Tract of Neonatal Calves.

Respiratory infections remain the second most common cause of clinical disease and mortality in newborn calves, which has led to increased interest in using vaccines early in life to mitigate this risk. Intranasal vaccination of neonatal calves can be an effective strategy to circumvent vaccine interference by maternal antibody, but this raises questions regarding onset of immune competence in the upper respiratory tract (URT) following birth. Little is known, however, about the development and function of mucosa-associated lymphoid tissue (MALT) in the URT of newborn calves and what factors, including the commensal microbiome, contribute to this early development. We review the structure, development, and function of MALT in the bovine URT during the first six weeks of life and identify knowledge gaps regarding this early developmental time. This information is critical when designing vaccination programs for young calves, especially when targeting respiratory pathogens that may reside within the commensal microbiome.