Bonnie Vecchiarelli

Temporal dynamics in the ruminal microbiome of dairy cows during the transition period.

The transition period in dairy cows refers to the period from 3 wk before calving to 3 wk post-calving and is a critical time for influencing milk production and cow health. We hypothesize that the ruminal microbiome shifts as dairy cows transition from a non-lactation period into lactation due to changes in dietary regimen. The purpose of this study was to identify differences in the ruminal microbiome of primiparous and multiparous (study group) cows during the transition period. Five primiparous and 5 multiparous cows were randomly selected from a herd, and ruminal contents were sampled, via stomach tube, 4 times (study day) at 3 wk before calving date (S1), 1 to 3 d post-calving (S2), and 4 (S3) and 8 wk (S4) into lactation and were evaluated for bacterial diversity using 16S pyrotags. Both groups received the same pre-fresh diet (14.6% CP, 44.0% NDF, 21.9% starch) and 3 different lactation diets (L1, L2, and L3) varying in forage base but not amount and formulated to have similar nutrient specifications (16.8% to 17.7% CP; 32.5% to 33.6% NDF; 26.2% to 29.1% starch) post-calving. Forty bacterial communities were analyzed on the basis of annotations of 100,000 reads, resulting in 15,861 operational taxonomic units grouped into 17 bacterial phyla. The UniFrac distance metric revealed that both study group and study day had an effect on the community compositions (P < 0.05; permutational multivariate ANOVA test). The most abundant phyla observed were Bacteroidetes and Firmicutes across all the communities. As the cows transitioned into lactation, the ratio of Bacteroidetes to Firmicutes increased from 6:1 to 12:1 (P < 0.05; Mann-Whitney U test), and this ratio was greater in primiparous cows than in multiparous cows (P < 0.05). This report is the first to explore the effect of parity on dynamics in the ruminal microbiome of cows during the transition period.

Associative patterns among anaerobic fungi, methanogenic archaea, and bacterial communities in response to changes in diet and age in the rumen of dairy cows.

The rumen microbiome represents a complex microbial genetic web where bacteria, anaerobic rumen fungi (ARF), protozoa and archaea work in harmony contributing to the health and productivity of ruminants. We hypothesized that the rumen microbiome shifts as the dairy cow advances in lactations and these microbial changes may contribute to differences in productivity between primiparous (first lactation) and multiparous (≥second lactation) cows. To this end, we investigated shifts in the ruminal ARF and methanogenic communities in both primiparous (n = 5) and multiparous (n = 5) cows as they transitioned from a high forage to a high grain diet upon initiation of lactation. A total of 20 rumen samples were extracted for genomic DNA, amplified using archaeal and fungal specific primers, sequenced on a 454 platform and analyzed using QIIME. Community comparisons (Bray–Curtis index) revealed the effect of diet (P < 0.01) on ARF composition, while archaeal communities differed between primiparous and multiparous cows (P < 0.05). Among ARF, several lineages were unclassified, however, phylum Neocallimastigomycota showed the presence of three known genera. Abundance of Cyllamyces and Caecomyces shifted with diet, whereas Orpinomyces was influenced by both diet and age. Methanobrevibacter constituted the most dominant archaeal genus across all samples. Co-occurrence analysis incorporating taxa from bacteria, ARF and archaea revealed syntrophic interactions both within and between microbial domains in response to change in diet as well as age of dairy cows. Notably, these interactions were numerous and complex in multiparous cows, supporting our hypothesis that the rumen microbiome also matures with age to sustain the growing metabolic needs of the host. This study provides a broader picture of the ARF and methanogenic populations in the rumen of dairy cows and their co-occurrence implicates specific relationships between different microbial domains in response to diet and age.

Metagenomic assessment of the functional potential of the rumen microbiome in Holstein dairy cows.

The microbial ecology of the rumen microbiome is influenced by the diet and the physiological status of the dairy cow and can have tremendous influence on the yield and components of milk. There are significant differences in milk yields between first and subsequent lactations of dairy cows, but information on how the rumen microbiome changes as the dairy cow gets older has received little attention. We characterized the rumen microbiome of the dairy cow for phylogeny and functional pathways by lactation group and stage of lactation using a metagenomics approach. Our findings revealed that the rumen microbiome was dominated by Bacteroidetes (70%), Firmicutes (15-20%) and Proteobacteria (7%). The abundance of Firmicutes and Proteobacteria were independently influenced by diet and lactation. Bacteroidetes contributed to a majority of the metabolic functions in first lactation dairy cows while the contribution from Firmicutes and Proteobacteria increased incrementally in second and third lactation dairy cows. We found that nearly 70% of the CAZymes were oligosaccharide breaking enzymes which reflect the higher starch and fermentable sugars in the diet. The results of this study suggest that the rumen microbiome continues to evolve as the dairy cow advances in lactations and these changes may have a significant role in milk production.

Metagenomic Analysis of the Rumen Microbiome of Steers with Wheat-Induced Frothy Bloat.

Frothy bloat is a serious metabolic disorder that affects stocker cattle grazing hard red winter wheat forage in the Southern Great Plains causing reduced performance, morbidity, and mortality. We hypothesize that a microbial dysbiosis develops in the rumen microbiome of stocker cattle when grazing on high quality winter wheat pasture that predisposes them to frothy bloat risk. In this study, rumen contents were harvested from six cannulated steers grazing hard red winter wheat (three with bloat score “2” and three with bloat score “0”), extracted for genomic DNA and subjected to 16S rDNA and shotgun sequencing on 454/Roche platform. Approximately 1.5 million reads were sequenced, assembled and assigned for phylogenetic and functional annotations. Bacteria predominated up to 84% of the sequences while archaea contributed to nearly 5% of the sequences. The abundance of archaea was higher in bloated animals (P < 0.05) and dominated by Methanobrevibacter. Predominant bacterial phyla were Firmicutes (65%), Actinobacteria (13%), Bacteroidetes (10%), and Proteobacteria (6%) across all samples. Genera from Firmicutes such as Clostridium, Eubacterium, and Butyrivibrio increased (P < 0.05) while Prevotella from Bacteroidetes decreased in bloated samples. Co-occurrence analysis revealed syntrophic associations between bacteria and archaea in non-bloated samples, however; such interactions faded in bloated samples. Functional annotations of assembled reads to Subsystems database revealed the abundance of several metabolic pathways, with carbohydrate and protein metabolism well represented. Assignment of contigs to CaZy database revealed a greater diversity of Glycosyl Hydrolases dominated by oligosaccharide breaking enzymes (>70%) in non-bloated samples. However, the abundance and diversity of CaZymes were greatly reduced in bloated samples indicating the disruption of carbohydrate metabolism. We conclude that mild to moderate frothy bloat results from tradeoffs both within and between microbial domains due to greater competition for substrates that are of limited availability as a result of biofilm formation.

A comparison of rumen microbial profiles in dairy cows as retrieved by 454 Roche and Ion Torrent (PGM) sequencing platforms

Next generation sequencing (NGS) technology is a widely accepted tool used by microbial ecologists to explore complex microbial communities in different ecosystems. As new NGS platforms continue to become available, it becomes imperative to compare data obtained from different platforms and analyze their effect on microbial community structure. In the present study, we compared sequencing data from both the 454 and Ion Torrent (PGM) platforms on the same DNA samples obtained from the rumen of dairy cows during their transition period. Despite the substantial difference in the number of reads, error rate and length of reads among both platforms, we identified similar community composition between the two data sets. Procrustes analysis revealed similar correlations (M2 = 0.319; P = 0.001) in the microbial community composition between the two platforms. Both platforms revealed the abundance of the same bacterial phyla which were Bacteroidetes and Firmicutes; however, PGM recovered an additional four phyla. Comparisons made at the genus level by each platforms revealed differences in only a few genera such as Prevotella, Ruminococcus, Succiniclasticum and Treponema (p < 0.05; chi square test). Collectively, we conclude that the output generated from PGM and 454 yielded concurrent results, provided stringent bioinformatics pipelines are employed.

Dysbiosis of the Fecal Microbiota in Cattle Infected with Mycobacterium avium subsp. paratuberculosis.

Johnes disease (JD) is a chronic, intestinal infection of cattle, caused by Mycobacterium avium subsp. paratuberculosis (MAP). It results in granulomatous inflammation of the intestinal lining, leading to malabsorption, diarrhea, and weight loss. Crohn’s disease (CD), a chronic, inflammatory gastrointestinal disease of humans, has many clinical and pathologic similarities to JD. Dysbiosis of the enteric microbiota has been demonstrated in CD patients. It is speculated that this dysbiosis may contribute to the intestinal inflammation observed in those patients. The purpose of this study was to investigate the diversity patterns of fecal bacterial populations in cattle infected with MAP, compared to those of uninfected control cattle, using phylogenomic analysis. Fecal samples were selected to include samples from 20 MAP-positive cows; 25 MAP-negative herdmates; and 25 MAP-negative cows from a MAP-free herd. The genomic DNA was extracted; PCR amplified sequenced on a 454 Roche platform, and analyzed using QIIME. Approximately 199,077 reads were analyzed from 70 bacterial communities (average of 2,843 reads/sample). The composition of bacterial communities differed between the 3 treatment groups (P < 0.001; Permanova test). Taxonomic assignment of the operational taxonomic units (OTUs) identified 17 bacterial phyla across all samples. Bacteroidetes and Firmicutes constituted more than 95% of the bacterial population in the negative and exposed groups. In the positive group, lineages of Actinobacteria and Proteobacteria increased and those of Bacteroidetes and Firmicutes decreased (P < 0.001). Actinobacteria was highly abundant (30% of the total bacteria) in the positive group compared to exposed and negative groups (0.1–0.2%). Notably, the genus Arthrobacter was found to predominate Actinobacteria in the positive group. This study indicates that MAP-infected cattle have a different composition of their fecal microbiota than MAP-negative cattle.

Alterations in ruminal bacterial populations at induction and recovery from diet-induced milk fat depression in dairy cows

Ten ruminally cannulated Holstein cows were used in a crossover design that investigated changes in ruminal bacterial populations in response to induction and recovery from diet-induced milk fat depression (MFD). Further, the effect on the ruminal microbiota of the cows with diet-induced milk fat depression inoculated with rumen contents from non-milk fat-depressed donor cows was evaluated. Milk fat depression was induced during the first 10 d of each period by feeding a low-fiber, high-starch, and high-polyunsaturated fatty acid diet (26.1% neutral detergent fiber, 28.1% starch, 5.8% total fatty acids, and 1.9% C18:2), resulting in a 30% decrease in milk fat yield. Induction was followed by a recovery phase, where all cows were switched to a high-fiber, low-starch, and low-polyunsaturated fatty acid diet (31.8% neutral detergent fiber, 23% starch, 4.2% total fatty acids, and 1.2% C18:2) and were allocated to (1) control (no inoculation) or (2) ruminal inoculation with donor cow digesta (8 kg/d for 6 d). Ruminal samples were collected at the end of induction (d 10) and during recovery (d 13, 16, and 28), separated to solid and liquid fractions, extracted for DNA, PCR- amplified for the V1-V2 region of the 16S rRNA gene, and analyzed for bacterial diversity. Results indicated that bacterial communities were different between fractions. In each fraction, differences were significant between the induction (d 10) and recovery (d 13, 16, and 28) periods; however, differences were less apparent with time during the recovery period. The MFD (d 10) was typified by a reduction in the relative sequence abundance of Bacteroidetes and an increase in the relative sequence abundance of Firmicutes and Actinobacteria across both fractions. At the genus level, relative sequence abundance of unclassified Lachnospiraceae, Butyrivibrio, Bulleidia, and Coriobacteriaceae were higher on d 10 and were positively correlated with trans-10,cis-12 CLA and the trans-10 isomer, suggesting their potential role in altered biohydrogenation reactions. A switch to the recovery diet resulted in a sharp increase in the Bacteroidetes lineages and a decrease in Firmicutes members on d 13; however, this shift appears to stabilize by d 28, indicating the restoration process for ruminal bacteria from an altered state is gradual and complex. Inoculation of 10% of rumen contents from non-MFD donor cows to MFD cows revealed this procedure had transient effects on only a few bacterial populations, and such effects disappeared after d 16 following cessation of inoculation. It can be concluded that alterations in milk FA profiles at induction are preceded by microbial alterations in the rumen driven by dietary changes.

Symposium review: Understanding diet–microbe interactions to enhance productivity of dairy cows

Ruminants are dependent on the microbiota (bacteria, protozoa, archaea, and fungi) that inhabit the reticulo-rumen for digestion of feedstuffs. Nearly 70% of energy and 50% of protein requirements for dairy cows are met by microbial fermentation in the rumen, emphasizing the need to characterize the role of microbes in feed breakdown and nutrient utilization. Over the past 2 decades, next-generation sequencing technologies have allowed for rapid expansion of knowledge concerning microbial populations and alterations in response to forages, concentrates, supplements, and probiotics in the rumen. Advances in gene sequencing and emerging bioinformatic tools have allowed for increased throughput of data to aid in our understanding of the functional relevance of microbial genomes. In particular, metagenomics can identify specific genes involved in metabolic pathways, and metatranscriptomics can describe the transcriptional activity of microbial genes. These powerful approaches help untangle the complex interactions between microbes and dietary nutrients so that we can more fully understand the physiology of feed digestion in the rumen. Application of genomics-based approaches offers promise in unraveling microbial niches and respective gene repertoires to potentiate fiber and nonfiber carbohydrate digestion, microbial protein synthesis, and healthy biohydrogenation. New information on microbial genomics and interactions with dietary components will more clearly define pathways in the rumen to positively influence milk yield and components.

Differences in the equine faecal microbiota between horses presenting to a tertiary referral hospital for colic compared with an elective surgical procedure

BACKGROUND The faecal microbiota is emerging as potentially important in intestinal disease. More research is needed to characterise the faecal microbiota from horses with colic. OBJECTIVES To compare the relative abundance of bacterial populations comprising the faecal microbiota in horses presenting for colic compared with an elective surgical procedure. STUDY DESIGN Prospective observational clinical study. METHODS Admission faecal samples were collected from horses presenting for colic and elective surgical procedures. Faecal samples were extracted for genomic DNA, PCR- amplified, sequenced and analysed using QIIME. Species richness and Shannon diversity were estimated for each faecal sample. The extent of the relationship between bacterial communities (beta diversity) was quantified using pairwise UniFrac distances, visualised using principal coordinate analysis (PCoA) and statistically analysed using PERMANOVA. The relative abundance of bacterial populations between the two treatment groups were compared using ANCOM. RESULTS Faecal bacterial communities in horses presenting for colic had fewer species (P<0.001) and lower diversity (P<0.001) compared with horses presenting for elective surgical procedures. Based on the PERMANOVA analysis, there was a significant difference in the bacterial community composition between horses admitted for colic vs. elective procedures (P = 0.001). Based on ANCOM test, at the genus level, 14 bacterial lineages differed between the two groups. The relative abundance of known commensal bacteria including Prevotella, Clostridia, Lachnospiraceae were reduced whereas Christenellaceae, Streptococcus and Sphaerochaeta were increased in horses with colic when compared with elective cases. MAIN LIMITATIONS Relative low numbers and a diverse population of horses. CONCLUSIONS Changes in bacterial populations in the faecal microbiota of horses presenting for colic observed in this study concurs with previous studies in veterinary and human patients with gastrointestinal disease. Future studies focusing on different causes of colic, chronic or recurrent disease, and the association with histological changes within the intestine are needed. The Summary is available in Portuguese - see Supporting Information.

Characterization of the fecal microbiota of healthy horses

OBJECTIVE To characterize the fecal microbiota of horses and to investigate alterations in that microbiota on the basis of sample collection site (rectum vs stall floor), sample location within the fecal ball (center vs surface), and duration of environmental exposure (collection time). ANIMALS 6 healthy adult mixed-breed mares. PROCEDURES From each horse, feces were collected from the rectum and placed on a straw-bedded stall floor. A fecal ball was selected for analysis immediately after removal from the rectum and at 0 (immediately), 2, 6, 12, and 24 hours after placement on the stall floor. Approximately 250 mg of feces was extracted from the surface and center of each fecal ball, and genomic DNA was extracted, purified, amplified for the V1-V2 hypervariable region of the 16S rDNA gene, and analyzed with a bioinformatics pipeline. RESULTS The fecal microbiota was unique for each horse. Bacterial community composition varied significantly between center and surface fecal samples but was not affected by collection time. Bacterial community composition varied rapidly for surface fecal samples. Individual bacterial taxa were significantly associated with both sample location and collection time but remained fairly stable for up to 6 hours for center fecal samples. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that, for horses, fecal samples for microbiota analysis should be extracted from the center of fecal balls collected within 6 hours after defecation. Samples obtained up to 24 hours after defecation can be analyzed with the realization that some bacterial populations may deviate from those immediately after defecation.