Nilusha Malmuthuge

Taxonomic Identification of Commensal Bacteria Associated with the Mucosa and Digesta throughout the Gastrointestinal Tracts of Preweaned Calves

ABSTRACT 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.

Regional and age dependent changes in gene expression of Toll-like receptors and key antimicrobial defence molecules throughout the gastrointestinal tract of dairy calves.

The primary aim of this study was to determine regional and age-dependent expression patterns of Toll-like receptors (TLRs), peptidoglycan recognition protein 1 (PGLYRP1), and β-defensin in rumen, jejunum, ileum, cecum and colon of 3 week (n=8) and 6 month old (n=8) calves. The expression of most TLRs was significantly down-regulated throughout the gastrointestinal tract (GIT) with increasing age. TLR10 expression was significantly higher in ileum than all other gut regions, irrespective of age. TLR2 and TLR4 expression were significantly higher in the cecum and colon of 6 month old calves. Furthermore, expression of β-defensin, and PGLYRP1 was only detectable in 6 month old calves. The expression of TLRs was positively or negatively correlated with population of total bacteria and/or lactic acid bacteria depending on the GIT region. These observations indicate that innate immune responses to commensal microflora may vary significantly throughout the GIT and with age changes.

Potential Regulatory Role of MicroRNAs in the Development of Bovine Gastrointestinal Tract during Early Life

This study aimed to investigate the potential regulatory role of miRNAs in the development of gastrointestinal tract (GIT) during the early life of dairy calves. Rumen and small intestinal (mid-jejunum and ileum) tissue samples were collected from newborn (30 min after birth; n = 3), 7-day-old (n = 6), 21-day-old (n = 6), and 42-day-old (n = 6) dairy calves. The miRNA profiling was performed using Illumina RNA-sequencing and the temporal and regional differentially expressed miRNAs were further validated using qRT-PCR. Analysis of 16S rRNA gene copy numbers was used to quantify total bacteria, Bifidobacterium and Lactobacillus species. The expression of miR-143 was abundant in all three gut regions, at all time points and it targets genes involved primarily in the proliferation of connective tissue cells and muscle cells, suggesting a role in regulating rapid tissue development during the early life of calves. The expression of miR-146, miR-191, miR-33, miR-7, miR-99/100, miR-486, miR-145, miR-196 and miR-211 displayed significant temporal differences (FDR <0.05), while miR-192/215, miR-194, miR-196, miR-205 and miR-31 revealed significant regional differences (FDR <0.05). The expression levels of miR-15/16, miR-29 and miR-196 were positively correlated with the copy numbers of 16S rRNA gene of Bifidobacterium or Lactobacillus species or both (P<0.05). Functional analysis using Ingenuity Pathway Analysis identified the above mentioned differentially expressed miRNAs as potential regulators of gut tissue cell proliferation and differentiation. The bacterial density-associated miRNAs were identified as modulators of the development of lymphoid tissues (miR-196), maturation of dendritic cells (miR-29) and development of immune cells (miR-15/16). The present study revealed temporal and regional changes in miRNA expression and a correlation between miRNA expression and microbial population in the GIT during the early life, which provides further evidence for another mechanism by which host-microbial interactions play a role in regulating gut development.

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 β-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.

Distinct commensal bacteria associated with ingesta and mucosal epithelium in the gastrointestinal tracts of calves and chickens

The primary aim of this study was to determine whether distinct gastrointestinal tract (GIT) microbial communities are established within ingesta and on mucosal surfaces of dairy calves and chickens to evaluate whether the principle of microbial segregation is of broad biological significance. Multivariate analysis of the predominant bacterial PCR-denaturing gradient gel electrophoresis profiles and estimated bacterial populations were compared in rumen, jejunum, ileum, cecum, and colon ingesta and matching mucosal tissues. Samples collected from 3-week old (n = 8) and 6-month old (n = 8) calves revealed that the predominant mucosa-associated bacteria were distinct from those inhabiting ingesta, and bacterial diversity varied significantly among the GIT regions. The estimated bacterial populations displayed significant regional differences for bovine mucosal (P = 0.05) and for ingesta (P = 0.03) only at 6 months of age. This indicates an established segregation of the enteric bacterial population throughout the GIT in weaned calves. Analysis of ileal and cecal bacterial profiles in chickens confirmed that the segregation of commensal bacteria between ingesta and the mucosal tissue was a common biological phenomenon. Our study provides some fundamental understanding of the impact of sample type (mucosa vs. ingesta), region, and host age on commensal bacterial establishment and segregation throughout the GIT.

The Gut Microbiome and Its Potential Role in the Development and Function of Newborn Calf Gastrointestinal Tract

A diverse microbial population colonizes the sterile mammalian gastrointestinal tract during and after the birth. There is increasing evidence that this complex microbiome plays a crucial role in the development of the mucosal immune system and influences newborn health. Microbial colonization is a complex process influenced by a two-way interaction between host and microbes and a variety of external factors, including maternal microbiota, birth process, diet, and antibiotics. Following this initial colonization, continuous exposure to host-specific microbes is not only essential for development and maturation of the mucosal immune system but also the nutrition and health of the animal. Thus, it is important to understand host–microbiome interactions within the context of individual animal species and specific management practices. Data is now being generated revealing significant associations between the early microbiome, development of the mucosal immune system, and the growth and health of newborn calves. The current review focuses on recent information and discusses the limitation of current data and the potential challenges to better characterizing key host-specific microbial interactions. We also discuss potential strategies that may be used to manipulate the early microbiome to improve production and health during the time when newborn calves are most susceptible to enteric disease.

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.

Understanding host-microbial interactions in rumen: searching the best opportunity for microbiota manipulation.

Ruminants utilize a wide variety of dietary substrates that are not digestible by the mammals, through microbial fermentation taking place in the rumen. Recent advanced molecular based approaches have allowed the characterization of rumen microbiota and its compositional changes under various treatment conditions. However, the knowledge is still limited on the impacts of variations in the rumen microbiota on host biology and function. This review summarizes the information to date on host-microbial interactions in the rumen and how we can apply such information to seek the opportunities to enhance the animal performance through manipulating the rumen function.

Model systems to analyze the role of miRNAs and commensal microflora in bovine mucosal immune system development

Information is rapidly accumulating regarding the role of miRNAs as key regulators of immune system development and function. It is also increasingly evident that miRNAs play an important role in host-pathogen interactions through regulation of both innate and acquired immune responses. Little is known, however, about the specific role of miRNAs in regulating normal development of the mucosal immune system, especially during the neonatal period. Furthermore, there is limited knowledge regarding the possible role the commensal microbiome may play in regulating mucosal miRNAs expression, although evidence is emerging that a variety of enteric pathogens influence miRNA expression. The current review focuses on recent information that miRNAs play an important role in regulating early development of the bovine mucosal immune system. A possible role for the commensal microbiome in regulating mucosal development by altering miRNA expression is also discussed. Finally, we explore the potential advantages of using the newborn calf as a model to determine how interactions between developmental programming, maternal factors in colostrum, and colonization of the gastrointestinal tract by commensal bacteria may alter mucosal miRNA expression and immune development. Identifying the key factors that regulate mucosal miRNA expression is critical for understanding how the balance between protective immunity and inflammation is maintained to ensure optimal gastrointestinal tract function and health of the whole organism.

Understanding the gut microbiome of dairy calves: Opportunities to improve early-life gut health1

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.