Devin B. Holman

The nasopharyngeal microbiota of feedlot cattle

The bovine nasopharyngeal tract plays an important role in animal health and welfare by acting as a site for the carriage of pathogens causing bovine respiratory disease, a condition which results in significant morbidity and mortality in feedlot cattle. We characterized the bacterial nasopharyngeal microbiota in cattle at feedlot entry (day 0) and day 60 using 454 pyrosequencing. We also identified the most frequently isolated aerobic bacteria from nasopharyngeal swabs after plating onto three types of media. The cattle nasopharyngeal microbiota was composed primarily of Proteobacteria (68.9%) and Firmicutes (19.2%). At the genus-level, there was more inter-individual variability and a total of 55 genera were identified. The genera Pseudomonas (23.7%), Shewanella (23.5%), Acinetobacter (17.5%), and Carnobacterium (12.2%) were most prevalent at entry, while after 60 days in the feedlot, Staphylococcus (20.8%), Mycoplasma (14.9%), Mannheimia (10.4%), and Moraxella (9.4%) were dominant. The nasopharyngeal microbiota also became more homogenous after 60 days in the feedlot and differed in structure at day 0 and 60. Using culture-based methods, the most frequently isolated bacteria from nasopharyngeal swabs were Bacillus, Staphylococcus, Moraxella, Pasteurella, and Mannheimia. These results provide insight into the nasopharyngeal microbiota of cattle and demonstrate that specific changes take place during feedlot production.

The nasopharyngeal microbiota of feedlot cattle that develop bovine respiratory disease.

Bovine respiratory disease is the major cause of morbidity and mortality in feedlot cattle. The objective of this study was to compare the nasopharyngeal bacterial microbiota of healthy cattle and cattle treated for BRD in a commercial feedlot setting using a high-density 16S rRNA gene microarray (Phylochip). Samples were taken from both groups of animals (n=5) at feedlot entry (day 0) and ≥60 days after placement. Cattle diagnosed with BRD had significantly less bacterial diversity and fewer OTUs in their nasopharynx at both sampling times. The predominant phyla in both groups were Proteobacteria and Firmicutes. The relative abundance of the phylum Actinobacteria was lower in cattle treated for BRD. At the family-level there was a greater relative abundance (P<0.05) of Micrococcaceae (day 0 only), Lachnospiraceae (≥60 days), Lactobacillaceae (day 0), and Bacillaceae (day 0) in healthy cattle compared to BRD-affected cattle. The community structure of the BRD-affected and healthy cattle were also significantly different from each other at both sampling times as measured using unweighted UniFrac distances. All entry samples of cattle diagnosed with BRD had 16S rRNA gene sequences representative of the BRD-associated bacteria Mannheimia haemolytica or Pasteurella multocida, although 3/5 healthy cattle were also positive for M. haemolytica at this time point. The results also indicate that the bovine nasopharyngeal microbiota is relatively unstable during the first 60 days in the feedlot.

Evolution of the nasopharyngeal microbiota of beef cattle from weaning to 40 days after arrival at a feedlot

Bovine respiratory disease complex (BRDc) is a major cause of morbidity and mortality in beef cattle. There is recent evidence suggesting that the nasopharyngeal microbiota has a key role in respiratory health and disease susceptibility in cattle. However, there is a paucity of knowledge regarding evolution of the nasopharyngeal microbiota when cattle are most likely to develop BRDc (i.e., from weaning to 40days after arrival at a feedlot). The objective was to describe the evolution of the nasopharyngeal microbiota of beef cattle from weaning to 40days after arrival at a feedlot. Deep nasal swabs (DNS) from 30 Angus-cross steers were collected at weaning, on arrival at a feedlot, and at day 40 after arrival. The DNA was extracted from DNS and the hypervariable region V3 of the 16S rRNA gene was amplified and sequenced (Illumina MiSeq platform). Nasopharyngeal microbiota underwent a profound evolution from weaning to arrival at the feedlot and from arrival to day 40, with the abundance of 92 Operational Taxonomic Units (OTUs) significantly changing over time. Mycoplasma (M. dispar and M. bovirhinis) was the most abundant genus in the nasopharynx, accounting for 53% of the total bacterial population. Because an evolving bacterial community may be less capable of resisting colonization by pathogenic bacteria, the instability of the nasopharyngeal microbiota documented in this study might explain why cattle are most likely to be affected with BRDc during the first weeks after weaning and arrival at a feedlot.

Effect of Co-Composting Cattle Manure with Construction and Demolition Waste on the Archaeal, Bacterial, and Fungal Microbiota, and on Antimicrobial Resistance Determinants.

Agricultural operations generate large quantities of manure which must be eliminated in a manner that is consistent with public health guidelines. Meanwhile, construction and demolition waste makes up about 25% of total solid municipal waste. Co-composting of manure with construction and demolition waste offers a potential means to make manure safe for soil amendment and also divert construction and demolition waste from municipal landfills. Therefore, the archaeal, bacterial, and fungal microbiota of two different types of composted cattle manure and one co-composted with construction and demolition waste, were assessed over a 99-day composting period. The microbiota of the three compost mixtures did not differ, but significant changes over time and by sampling depth were observed. Bacillus and Halocella, however, were more relatively abundant in composted manure from cattle fed dried distillers’ grains and solubles. Proteobacteria and Bacteroidetes were enriched at day 0 and Firmicutes at day 99. The fungal genus Kernia was the most relatively abundant overall and was enriched at day 0. The concentration of 12 antimicrobial resistance determinants in the compost mixtures was also determined, and 10 of these determinants decreased significantly from days 0 to 99. The addition of construction and demolition waste did not affect the persistence of antimicrobial resistance genes or community structure of the compost microbiota and therefore co-composting construction and demolition waste with cattle manure offers a safe, viable way to divert this waste from landfills.

Injectable antimicrobials in commercial feedlot cattle and their effect on the nasopharyngeal microbiota and antimicrobial resistance

Beef cattle in North America that are deemed to be at high risk of developing bovine respiratory disease (BRD) are frequently administered a metaphylactic antibiotic injection to control the disease. Cattle may also receive in-feed antimicrobials to prevent specific diseases and ionophores to improve growth and feed efficiency. Presently, attempts to evaluate the effects that these medications have on antibiotic resistance in the bovine nasopharyngeal microbiota have been focused on culturable bacteria that are associated with BRD. Therefore, we assessed the effects of injectable antibiotics on the nasopharyngeal microbiota of commercial feedlot cattle in Alberta, Canada, through the first 60 d on feed. Although all cattle in the study were also receiving in-feed chlortetracycline and monensin, the administration of a single injection of either oxytetracycline or tulathromycin at feedlot placement altered the nasopharyngeal microbiota in comparison with the cattle receiving only in-feed antibiotics. Oxytetracycline significantly (P < 0.05) reduced the relative abundance of Mannheimia spp. from feedlot entry to exit (≥60 d) and both oxytetracycline and tulathromycin treated cattle had a significantly lower relative abundance of Mycoplasma spp. at feedlot exit compared with the in-feed antibiotic only group. The proportion of the tetracycline resistance gene tet(H) was significantly increased following oxytetracycline injection (P < 0.05). Oxytetracycline also reduced both the number of OTUs and the Shannon diversity index in the nasopharyngeal microbiota (P < 0.05). These results demonstrate that in feedlot cattle receiving subtherapeutic in-feed antimicrobials, the administration of a single injection of either oxytetracycline or tulathromycin resulted in measurable changes to the nasopharyngeal microbiota during the first 60 d following feedlot placement.

Incubation Temperature, But Not Pequi Oil Supplementation, Affects Methane Production, and the Ruminal Microbiota in a Rumen Simulation Technique (Rusitec) System

Lipid supplementation is a promising strategy for methane mitigation in cattle and has been evaluated using several different lipid sources. However, limited studies have assessed the effect of temperature on methane emissions from cattle and changes in incubation temperature have also not been extensively evaluated. The aim of this study was to evaluate the combined effect of pequi oil (high in unsaturated fatty acids) and incubation temperature on fermentation characteristics and microbial communities using the rumen simulation technique. A completely randomized experiment was conducted over a 28-day period using a Rusitec system. The experiment was divided into four periods of 7 days each, the first of which was a 7-day adaptation period followed by three experimental periods. The two treatments consisted of a control diet (no pequi oil inclusion) and a diet supplemented with pequi oil (1.5 mL/day) which increased the dietary fat content to 6% (dry matter, DM-basis). Three fermenter vessels (i.e., replicates) were allocated to each treatment. In the first experimental period, the incubation temperature was maintained at 39°C, decreased to 35°C in the second experimental period and then increased again to 39°C in the third. Pequi oil was continuously supplemented during the experiment. Microbial communities were assessed using high-throughput sequencing of the archaeal and bacterial 16S rRNA gene. Methane production was reduced by 57% following a 4°C decrease in incubation temperature. Supplementation with pequi oil increased the dietary fat content to 6% (DM-basis) but did not affect methane production. Analysis of the microbiota revealed that decreasing incubation temperature to 35°C affected the archaeal and bacterial diversity and richness of liquid-associated microbes, but lipid supplementation did not change microbial diversity.

The type of forage substrate preparation included as substrate in a RUSITEC system affects the ruminal microbiota and fermentation characteristics

In vitro fermentation systems such as the rumen simulation technique (RUSITEC) are frequently used to assess dietary manipulations in livestock, thereby limiting the use of live animals. Despite being in use for nearly 40 years, improvements are continually sought in these systems to better reflect and mimic natural processes in ruminants. The aim of this study was to evaluate the effect of forage preparation, i.e., frozen minced (FM) and freeze-dried and ground (FDG), on the ruminal microbiota and on fermentation characteristics when included as a substrate in a RUSITEC system. A completely randomized design experiment was performed over a 15-day period, with 7 days of adaptation and an 8-day experimental period. Fermentation parameters (total gas, CH4, and volatile fatty acid production) were analyzed on a daily basis over the experimental period and the archaeal and bacterial microbiota (liquid-associated microbes [LAM] and solid-associated microbes [SAM] was assessed at 0, 5, 10, and 15 days using high-throughput sequencing of the 16S rRNA gene. Results from this study suggested a tendency (P = 0.09) of FM treatment to increase daily CH4 (mg/d) production by 16.7% when compared with FDG treatment. Of the major volatile fatty acids (acetate, propionate, and butyrate), only butyrate production was greater (P = 0.01) with FM treatment compared with FDG substrate. The archaeal and bacterial diversity and richness did not differ between the forage preparations, although feed particle size of the forage had a significant effect on microbial community structure in the SAM and LAM samples. The Bacteroidetes phylum was more relatively abundant in the FM substrate treatment, while Proteobacteria was enriched in the FDG treatment. At the genus-level, Butyrivibrio, Prevotella, and Roseburia were enriched in the FM substrate treatment and Campylobacter and Lactobacillus in the FDG substrate treatment. Evidence from this study suggests that forage preparation affects CH4 production, butyrate production, and the structure of the rumen microbiota during in vitro fermentation.

Changes in bacterial community composition of Escherichia coli O157:H7 super-shedder cattle occur in the lower intestine

Escherichia coli O157:H7 is a foodborne pathogen that colonizes ruminants. Cattle are considered the primary reservoir of E. coli O157:H7 with super-shedders, defined as individuals excreting > 104 E. coli O157:H7 CFU g-1 feces. The mechanisms leading to the super-shedding condition are largely unknown. Here, we used 16S rRNA gene pyrosequencing to examine the composition of the fecal bacterial community in order to investigate changes in the bacterial microbiota at several locations along the digestive tract (from the duodenum to the rectal-anal junction) in 5 steers previously identified as super-shedders and 5 non-shedders. The overall bacterial community structure did not differ by E. coli O157:H7 shedding status; but several differences in the relative abundance of taxa and OTUs were noted between the two groups. The genus Prevotella was most enriched in the non-shedders while the genus Ruminococcus and the Bacteroidetes phylum were notably enriched in the super-shedders. There was greater bacterial diversity and richness in samples collected from the lower- as compared to the upper gastrointestinal tract (GI). The spiral colon was the only GI location that differed in terms of bacterial diversity between super-shedders and non-shedders. These findings reinforced linkages between E. coli O157:H7 colonization in cattle and the nature of the microbial community inhabiting the digestive tract of super-shedders.

Tucumã Oil Shifted Ruminal Fermentation, Reducing Methane Production and Altering the Microbiome but Decreased Substrate Digestibility Within a RUSITEC Fed a Mixed Hay – Concentrate Diet

Tucumã oil is sourced from the fruit pulp of the tucumã tree and contains high concentrations of unsaturated fatty acids and carotenoids. Due to these properties it may have the potential to decrease enteric methane (CH4) from ruminants when included in the diet. The objective of this study was to determine the effect of oil mechanically extracted from the fruit pulp of tucumã on fermentation characteristics, CH4 production and the microbial community using the rumen stimulation technique. Treatments consisted of a control diet (forage:concentrate; 70:30), and tucumã oil included at 0.5 or 1.0% (v/v). Addition of tucumã oil linearly decreased (P < 0.01) dry matter disappearance. Total gas (mL/d) and carbon dioxide (CO2) production (mL/d, mL/g DM) were unaffected (P ≥ 0.36) to increasing addition of tucumã oil where 0.5% (v/v) of Tucumã oil numerically increased both variables. Acetate and butyrate percentages of total VFA were linearly decreased (P ≤ 0.01) and propionate and valerate percentages of total VFA were linearly increased (P < 0.01) by increasing concentrations of tucumã oil added to the substrate. The ratio of acetate to propionate was linearly decreased (P < 0.01) with increasing concentration of tucumã oil. Methane production (mL/d) was linearly decreased (P = 0.04) with increasing addition of tucumã oil to the substrate. Tucumã oil reduced the bacterial richness and diversity when included at 1.0% (v/v) in both solid- and liquid- associated microbes. The abundance of the genera Fibrobacter and Rikenellaceae RC9 gut group were decreased and Pyramidobacter, Megasphaera, Anaerovibrio, and Selenomonas were enriched by the addition of 1.0% tucumã oil. In conclusion, tucumã oil resulted in the favorable shift in fermentation products away from acetate toward propionate, decreasing the production of CH4 when tucumã oil was included at 1.0% (v/v), however, substrate digestibility was also inhibited. The rumen microbiota was also altered by the addition of tucumã oil.

Humic substances alter ammonia production and the microbial populations within a RUSITEC fed a mixed hay – concentrate diet

Humic substances are a novel feed additive which may have the potential to mitigate enteric methane (CH4) production from ruminants as well as enhance microbial activity in the rumen. The aim of this study was to examine the effects of humic substances on fermentation characteristics and microbial communities using the rumen stimulation technique (RUSITEC). The experiment was conducted as a completely randomized design with 3 treatments duplicated in 2 runs (a 15-day period each run) with 2 replicates per run. Treatments consisted of a control diet (forage:concentrate; 60:40) without humic substances or humic substances added at either 1.5 g/d or 3.0 g/d. Dry matter disappearance, pH, fermentation parameters and gas production were measured from day 8 to 15. Samples for microbial profiling were taken on day 5, 10, and 15 using the digested feed bags for solid- associated microbes (SAM) and fermenter fluid for liquid- associated microbes (LAM). The inclusion of humic substances had no effect (P ≥ 0.19) on DM disappearance, pH or the concentrations of VFA. The production of NH3 was linearly decreased (P = 0.04) with increasing levels of humic substances in the diet. There was no effect (P ≥ 0.43) of humic substances on total gas, CO2 or CH4 production. The number of OTUs was significantly reduced in the 3.0 g/d treatment compared to the control on d 10 and 15; however, the microbial community structure was largely unaffected (P > 0.05). In the SAM samples, the genera Lachnospiraceae XPB1014 group, Succiniclasticum, and Fibrobacter were reduced in the 3.0 g/d treatment and Anaeroplasma, Olsenella, and Pseudobutyrivibrio were increased on day 5, 10, and 15. Within the LAM samples, Christensenellaceae R-7 and Succiniclasticum were the most differentially abundant genera between the control and 3.0 g/d HS treatment samples (P < 0.05). This study highlights the potential use of humic substances as a natural feed additive which may play a role in nitrogen metabolism without negatively affecting the ruminal microbiota.