A metagenome expression library of bulk DNA extracted from the rumen content of a dairy cow was established in a phage lambda vector and activity-based screening employed to explore the functional diversity of the microbial flora. Twenty-two clones specifying distinct hydrolytic activities (12 esterases, nine endo-beta-1,4-glucanases and one cyclodextrinase) were identified in the library and characterized. Sequence analysis of the retrieved enzymes revealed that eight (36%) were entirely new and formed deep-branched phylogenetic lineages with no close relatives among known ester- and glycosyl-hydrolases. Bioinformatic analyses of the hydrolase gene sequences, and the sequences and contexts of neighbouring genes, suggested tentative phylogenetic assignments of the rumen organisms producing the retrieved enzymes. The phylogenetic novelty of the hydrolases suggests that some of them may have potential for new applications in biocatalysis.

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We conducted a lactation trial with a fresh forage diet in order to evaluate 1) the effects of monensin on nitrogen metabolism, and 2) the Cornell Net Carbohydrate and Protein System (CNCPS). Thirty Holstein cows in midlactation (eight fitted with ruminal fistulas) were gradually introduced to a fresh forage diet. A concentrate mix based on corn meal was fed before the a.m. and p.m. milking times 0730 and 1730 h, then the fresh forage was fed at 0830 and 1830 h. Fifteen cows each were allocated to a control (no monensin) and a treatment group receiving 350 mg/cow per day of monensin in the p.m. concentrate feeding. A 7-d fecal and urine collection period and a 3-d rumen sampling period were conducted with the fistulated cows. After the lactation study was concluded, the fistulated cows were fed forage regrowth and a 3-d rumen sampling period was repeated. Monensin increased milk production by 1.85 kg. Milk fat and protein concentrations decreased and milk fat and protein yields increased, but the effects were nonsignificant. Monensin did not significantly affect DMI. Ruminal ammonia and the acetate-to-propionate ratio decreased with the addition of monensin in both fed forages. Monensin decreased fecal N output, and increased apparent N digestibility by 5.4%. Because of the decrease in ruminal ammonia and increase in apparent N digestibility, we concluded monensin was sparing amino acids from wasteful rumen degradation with a fresh forage diet. The precision of the CNCPS in predicting performance was high (r2 = 0.76), and the bias was low (overprediction of 3.6%). These results indicate that the CNCPS can be used for dairy cows consuming fresh forage and gives realistic predictions of performance.

The internal transcribed spacer (ITS) is a popular barcode marker for fungi and in particular the ITS1 has been widely used for the anaerobic fungi (phylum Neocallimastigomycota). A good number of validated reference sequences of isolates as well as a large number of environmental sequences are available in public databases. Its highly variable nature predisposes the ITS1 for low level phylogenetics; however, it complicates the establishment of reproducible alignments and the reconstruction of stable phylogenetic trees at higher taxonomic levels (genus and above). Here, we overcame these problems by proposing a common core secondary structure of the ITS1 of the anaerobic fungi employing a Hidden Markov Model-based ITS1 sequence annotation and a helix-wise folding approach. We integrated the additional structural information into phylogenetic analyses and present for the first time an automated sequence-structure-based taxonomy of the ITS1 of the anaerobic fungi. The methodology developed is transferable to the ITS1 of other fungal groups, and the robust taxonomy will facilitate and improve high-throughput anaerobic fungal community structure analysis of samples from various environments.

To obtain information on the diversity of ruminal methanogens in grazing animals, three ruminal methanogens from grazing cattle were characterized and identified. Two of the isolates were rod-shaped, with one staining Gram-positive and being non-motile (BRM9), and the other (BRM16) staining Gram-negative and being motile. These isolates grew only on H(2)/CO(2) and formate, and optimally at 38 degrees C and pH 6.5-7.0. The third isolate (CM1) was non-motile, pseudosarcina-shaped, and grew on H(2)/CO(2), acetate, and methyl-containing compounds, with optimal growth at 40 degrees C and pH 6.5. DNA was prepared from the three isolates, and their 16S rRNA genes were sequenced. Phenotypic data and comparisons of nearly complete 16S rDNA sequences showed that BRM9, BRM16, and CM1 are strains of Methanobacterium formicicum, Methanomicrobium mobile, and Methanosarcina barkeri respectively. To the best of our knowledge, this is the first information on ruminal methanogens in cattle maintained under grazing management.

Escherichia coli O157:H7 is a pathogenic bacterium that causes acute illness in humans, but mature cattle are not affected. E. coli O157:H7 can enter the human food supply from cattle via fecal contamination of beef carcasses at slaughter. Previous attempts to correlate the incidence of E. coli O157:H7 with specific diets or feeding management practices gave few statistically significant or consistent findings. However, recent work indicates that cattle diets may be changed to decrease fermentation acid accumulation in the colon. When fermentation acids accumulate in the colon and pH decreases, the numbers of acid-resistant E. coli increase; acid-resistant E. coli are more likely to survive the gastric stomach of humans. When cattle were fed hay for a brief period (<7 d), acid-resistant E. coli numbers declined dramatically. Other workers have shown that brief periods of hay feeding can also decrease the number of cattle shedding E. coli O157:H7, and a similar trend was observed if cattle were taken off feed and exposed to simulated transport. These observations indicate that cattle feeding management practices may be manipulated to decrease the risk of foodborne illness from E. coli, but further work will be needed to confirm these effects.

Twenty-four multiparous and fifteen first lactation Holstein cows averaging 263 days in milk and weighing 614 kg were fed diets adequate or deficient in ruminal nitrogen (N), based on predictions of the Cornell Net Carbohydrate and Protein System (CNCPS). After adjustment to a low crude protein (CP) total mixed rations (TMR; 12.6% CP), the cows were allocated to 13 blocks based on lactation number, milk production, body condition score, and body weight. Within each block, cows were randomly assigned to one of the 3 treatment (TRT) diets (9.4, 11.1 and 14.1% CP for TRT 1, 2, and 3, respectively). All diets contained the same proportion of high moisture corn, chopped grass hay, and minerals, with urea substituted for corn silage as needed to reach the three CP levels. The TRT diets were then fed to the cows for 4 wk. Milk production was significantly affected by TRT: 15.5, 18.8, and 21.7 kg/d for TRT diets 1, 2, and 3, respectively. DMI was increased significantly as the percentage of CP increased from 9.4 to 14.1% CP: 17.6, 20.0, and 21.2 kg/d for TRT diets 1,2, and 3, respectively. CNCPS predictions for production (with and without the N adjustment for ruminal N deficiency) of metabolizable protein (MP) allowable milk were compared with observed milk production. Using the average individual weekly cow data from all 3 TRT, we found that the CNCPS accounted for 72 and 68% of the variation in MP allowable milk without and with the N deficiency adjustment, respectively. The overall mean bias without the N adjustment was 3.3 kg of milk (over prediction model bias of 14.6%, P < 0.001), and the N adjustment reduced the model over-prediction bias to 0.01 kg of milk (P = 0.96).

A procedure to eliminate Escherichia coli in dairy cattle manure was developed. E. coli persisted in fresh manure and farm storage tanks, and viable counts ranged from 105 to 108/g. If the feces to urine ratio of fresh manure was decreased from 2.2 to 1, E. coli did not persist for ≥10 days (<10 viable cells/g), and it appeared that the urine was killing E. coli. Fecal urease contamination produced CO2, and 16% was trapped as carbonate. When urine pH was decreased, antimicrobial effect was lost, even if the pH was readjusted to 8.5. When E. coli K-12 and O157:H7 were treated with Na2CO3 (100 mM, pH 8.5, 24 h), viable cells were not detected. The E. coli count of manure (feces to urine ratio of 2.2:1) was decreased by Na2CO3 addition (8 g/kg), but pH sometimes declined and carbonate was lost. When NaOH was included (2 g/kg), Na2CO3 additions could be decreased (4 g/kg), and treatment time was 5 days. Treatment cost could be <$10 year-1 (dairy cow)-1. Water dilution (3-fold) did not diminish the effectiveness of the carbonate/alkali treatment, and viability was <10 cells/g.