Progress 10/01/99 to 09/30/04
Outputs
The ability to describe microbial diversity is an important goal of microbial ecology. Historically, microbiologists have relied on culture-dependent methods but now culture-independent methods are available to explore diversity in complex environmental systems. One of these approaches is terminal-restriction fragment length polymorphism (T-RFLP) analysis of amplified ribosomal RNA genes which is used to monitor microbial diversity and for semi-quantitative estimates of species richness and relative abundance in environmental samples. Our goal has been to assess the utility of T-RFLP community profiles in representation of relative abundance of bacteria in intentional community samples using five species of ruminal bacteria. Template DNA for PCR amplification was generated by mixing equal quantities of genomic DNA from pure cultures or by mixing equal numbers of cells prior to DNA extraction. Pairwise mixtures of Fibrobacter succinogenes S85 with Ruminococcus albus 8,
Ruminococcus flavefaciens FD-1, Butyrivibrio fibrisolvens 49 and Streptococcus bovis JB1 were performed and a 5-member community was constructed. Genome size was known for 3 of the 5 bacterial species and the rRNA operon copy number was estimated for all species. Relative abundance calculations of T-RFs generated in genomic DNA mixes indicated that PCR product ratios, as determined by T-RFLP, did not reflect input ratios. This discrepancy could not be accounted for by differences either in genome size and rRNA operon copy number. Relative abundance calculations determined by T-RFLP analyses statistically differed between parallel genomic DNA and cell mixes. Cell mixes showed that the DNA extraction procedure was biased towards easily lysed cells, even if bead-beating was incorporated into the protocol. The dependence of T-RFLP peak ratios on DNA extraction and PCR amplification prevents accurate quantification of the relative abundance of microorganisms that are reflective of natural
populations. The results of this study indicate that although T-RFLP is a robust technique for describing differences in simple microbial communities, the ability of the technique to quantitatively describe the relative abundance of microorganisms in native communities is limited. Biases inherent to DNA extraction and PCR amplification also affect T-RFLP analyses and ratios of T-RFs generated are only reflective of PCR amplification product abundances, not of gene abundances in natural environments.
Impacts
Understanding how microbial populations change along the gastrointestinal tracts of ruminants in response to changes in diet and management sould make it more feasible to tailor ruminant diets to the requirements of the ruminal microbes as well as to needs of the host animals. Several practical outcomes would be achieved: 1) farmers could more precisely formulate rations to minimize environmentally detrimental nutrient excretion and 2) considerable savings in feeds costs to producers would be realized. We are applying these data to the development of a widely used computer model to predict animal performance and to quantify nutrient excretion with an emphasis on nitrogen and phosphorus. This model is used to assess and reduce the environmental impact of beef and dairy operations in New York State and elsewhere in the United States and internationally.
Publications
- Frey, J.C., Angert, E.R. and Pell, A.N. 2004. Ruminal Bacteria for the Assessment of Biases in Terminal-Restriction Fragment Length Polymorphism, A.S.M. Annual Meeting. May, 2004.
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Progress 01/01/03 to 12/31/03
Outputs
The goal of our project is to determine how microbial populations shift along the gastrointestinal tract of lactating dairy cows. We are using several molecular microbial ecology methods to monitor these changes in ruminal, abomasal and ileal samples as well as feces. We also have information on animal performance and diet composition from the experimental animals. Our long-term goal is to use these data to refine the Cornell Net Carbohydrate and Protein System. To this end, we have refined a DNA extraction procedure to obtain high molecular weight genomic DNA from five species of ruminal bacteria: Fibrobacter succinogenes S85, Ruminococcus albus 8, Ruminococcus flavefaciens FD1, Butyrivibrio fibrisolvens 49 and Streptococcus bovis JB1. This procedure which involves both enzyme treatment and mechanical lysis successfully isolated high molecular weight genomic DNA from both the Gram positive and Gram negative organisms. More interesting, the method extracts large
amounts of plasmid DNA from all species except F. succinogenes S85. To confirm this hypothesis, the lower MW bands from the extraction of S. bovis JB1 were isolated and cloned. Clones shared 100 percent identity with the previously reported S. bovis JB1 plasmid pSOB2. These data confirm that the optimized method is a valuable extraction procedure for obtaining both genomic DNA and for the extraction of plasmid DNA from target organisms of interest. This year we completed our series of experiments using Southern hybridization to estimate the rRNA operon copy number of five ruminal microbes. The operon copy number of F. succinogenes S85, R. albus RA8, R. flavefaciens FD1, B. fibrisolvens 49 and S. bovis JB1 were estimated to be 3, 3, 3, 4 and 7, respectively. Experiments to investigate the effects of cell lysis efficiency and rRNA operon copy number on Terminal Restriction Fragment Length Polymorphism (T-RFLP) are underway. The T-RFLP technique provided accurate size calling with DNA
from pure cultures as all experimental T-RF values were within 1 base pair of the predicted values. Preliminary findings suggest that neither percent height nor percent area generated from the electropherograms can be used quantitatively. When equal numbers of cells per milliliter of S. bovis JB1 and R. flavefaciens FD1 were mixed together, S. bovis JB1 was over-represented and R. flavefaciens FD1 under-represented using percent area and percent height as criteria in the T-RFLP analysis.
Impacts
Understanding how microbial populations change along the gastrointestinal tract and in response to dietary change could make it more possible to tailor ruminant diets to the microbes as well as to the host animals. This could have several important practical implications: 1) producers could feed better-defined precision rations to minimize environmentally detrimental nutrient excretion and 2) more refined rations could result in considerable cost savings to producers. Because of the breadth of the data set with which we are working, we are hopeful that we will be able to refine the ruminal submodel of the Cornell Net Carbohydrate and Protein System.
Publications
- Arcuri, P.B., M.L. Thonney, P. Schofield and A.N. Pell. 2003. Polyethylene glycol and polyvinylpyrrolidone effects on bacterial rRNA extraction and hybridization from cells exposed to tannins. Brazilian Journal of Agricultural Research 38(9):1073-1081.
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Progress 01/01/02 to 12/31/02
Outputs
We have been involved in two research areas: 1) use of T-RFLP (terminal restriction fragment length polymorphism) analysis to study change in ruminal microbial populations, and 2) measurement of carbohydrate digestion rates. The goal for the microbial ecology research has been to follow changes in the populations of fiber-degrading bacteria to refine the Cornell Net Carbohydrate and Protein System (CNCPS) model. To date, we have 1) optimized DNA extraction of key rumen bacteria, 2) determined the rRNA operon number for each of these four fiber-digesting species, and 3) assessed the utility of T-RFLP in following cellulolytic diversity. The DNA extraction protocol was designed to maximize recovery of DNA from our indicator species. An initial heat treatment under anaerobic conditions reduced degradation of genomic DNA, perhaps by inactivating endogenous endonucleases released by the anaerobic microorganisms due to oxygen exposure. Cells were heated anaerobically at 65C
for 10 min, pelleted and resuspended in TE buffer. The cells were treated with lytic enzymes (lysozyme, mutanolysin, achromopeptidase and Proteinase K) and subjected to freeze-thawing. The DNA was further purified with CTAB and phenol-chloroform extraction. Many quantitative methods indirectly measure species abundance by quantifying genes encoding the small subunit rRNA. Depending on the species, a bacterial genome can harbor one to 15 copies of the rRNA operon so rRNA operon number can affect rDNA-based quantification methods. To assess the effect of rDNA copy number on these measurements, we estimated the rRNA operon number of our indicator species using southern hybridization. The rRNA operon number of F. succinogenes S85, R. albus RA8, B. fibrisolvens 49, S. bovis JB1 were estimated to be 3, 3, 4, 7, respectively. To assess the utility of T-RFLP analyses to track ruminal microbial diversity, pilot experiments with DNA from key cellulolytic bacteria and S. bovis were performed.
Universal primers, 5' 6-FAM (carboxyfluorescein) 515F and 1392R was used to amplify 16S rDNA from F. succinogenes S85, R. albus 8, R. flavefaciens FD1 and S. bovis JB1 thus labelling the 5' end of the amplification products. Purified PCR products were digested with MseI (New England Biolabs), mixed with GeneScan 500 TAMRA size standard and run on a sequencer. The GeneScan software package (Applied Biosystems) was used to determine the sizes of the labeled Terminal Restriction Fragments. The T-RFLP technique provided accurate size-calling with pure cultures as all experimental values were within 1 base pair of the predicted values. These experiments demonstrate the feasibility of using T-RFLP for ruminal community analysis. In our studies of digestion rates, we studied the use of near infrared spectroscopy (NIRS) to measure digestion rates of starch and fiber. The results from grain samples suggest that use of NIRS for prediction of digestion rates is possible and that much of the
noise in the calibration is due to variation in the reference methods. We have determined the digestion rates of the carbohydrate fractions of brown midrib forage sorghum.
Impacts
The microbiological research makes it possible to evaluate changes in the ruminal microbial populations which in turn dictate the efficiency with which ruminants use energy and protein. The microbiological and digestion rate data will be used to refine nutritional models to predict animal performance and the impact of livestock on the environment.
Publications
- Cannas, A, P.J. Van Soest, and A.N. Pell. 2002. Use of animal and dietary information to predict rumen turnover. Anim. Feed Sci. Technol. 103: 1-23.
- Tedeschi, L. O., D. G. Fox, A. N. Pell, D. P. Lanna, and C. Boin. 2002. Development and evaluation of a tropical feed library for the Cornell Net Carbohydrate and Protein System model. Scientia Agricola 59:1-18.
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Progress 01/01/01 to 12/31/01
Outputs
This year much of our effort has been devoted to 2 projects: 1) use of T-RFLP (terminal restriction fragment length polymorphism) analysis to study the fluctuations of microbial populations in the rumen, and 2) on-going studies on measurement of digestion rates of carbohydrates. For the T-RFLP work, extraction and PCR conditions have been optimized to minimize detection bias due to lysis, extraction and PCR amplification methods. The operon number of key bacterial species has been determined and preliminary experiments with pure cultures in known mixtures have been conducted. This research is in its preliminary stages with no publications yet. We have continued to study the contribution of organic acids and sugars to gas production and microbial yield. Clear differences in both amount and rate of gas production were detected between lactic, malic and acetic acids. Fermentation of citric acid yielded the most gas and had the highest acetate:propionate ratio although
not necessarily the highest rate. We also have begun to investigate the use of near-infrared spectroscopy for prediction of digestion rates of starch and fiber. The relationship between NIR spectra and starch digestion rate of dried corn, wheat, barley and milo samples was > 0.9.
Impacts
The new methods will yield data for use of Beef, Dairy NRC and other nutritional models. Knowledge of how diet changes ruminal microbial populations will permit more efficient use of feeds and less excretion.
Publications
- Clark, J.H., D.K. Beede, R.A. Erdman, J.P. Goff, R.R. Grummer, J.G. Linn, A.N. Pell, C.G. Schwab, T. Tomkins, G.A. Varga and W.P. Weiss. 2001. Nutrient Requirements of Dairy Cattle, 7th Revised Ed., National Academy Press. Washington, D.C.
- Tedeschi, L.O., A.N. Pell, D.G. Fox, and C.R. Llames. 2001. The amino acid profile of the whole plant and of four fractions from temperate and tropical forages. J. Anim. Sci. 79:525-532.
- Cannas A., D.G. Fox, A.N. Pell, P.J. Van Soest 2001. Adaptation of the Cornell Net Carbohydrate and Protein System to Sheep: Validation of feed digestibility. J. Anim. Sci. 79(Supp1):358.
- Molina, D.O., A. N. Pell, P. Schofield. 2001.Gas and VFA production during the in vitro fermentation of selected organic acids and sugars. J. Anim. Sci. 79(Supp1):79.
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Progress 01/01/00 to 12/31/00
Outputs
We continued to investigate the relationship between gas production, pH, substrate disappearance, volatile fatty acid production and microbial yield from fermentation of malate, citrate and lactate. In addition, we have submitted a paper comparing the amino acid profile of the whole forage with profiles of various fractions including the borate buffer soluble residue, the neutral detergent soluble residue and the acid detergent soluble residue. A range of temperate and tropical grasses and legumes were studied. With a few notable exceptions, the amino acid profiles of the extracted residues were similar to those from the unfractionated forage. Significant efforts were devoted to refining methods to measure digestion rates using gas production including modifications of the gas production methods and curve-fitting routines. Data from temperate and tropical forages were collected for inclusion in the feed library of the Cornell Net Carbohydrate and Protein System.
Preliminary data were collected to determine the NIR profiles of pure cultures of several species of ruminal bacteria including Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes and Prevotella ruminicola and mixed ruminal samples.
Impacts
Our improved feed analyses, part of development of the Cornell Net Carbohydrate and Protein system, a computer model that predicts nutrient excretion, can avert adverse environmental effects of ruminant livestock.
Publications
- Pell, A.N., Molina, D.O., and Schofield, P. 2000. Measurement of gas production in vitro. In Gas Production: Fermentation Kinetics for Feed Evaluation and to Assess Microbial Activity. BSAS Occasional Publication. pp. 1-12.
- Pell, A.N., Woolston, T.K., Nelson, K.E., and Schofield, P. 2000. Tannins: Biological activity and bacterial tolerance. In Tannins in Human and Animal Nutrition, pp. 111-117. Ed. J.D. Brooker. Aust. Ctr. Int. Agric. Res. (ACIAR) Proceedings No. 92, Adelaide, Australia.
- Pell, A.N., Wu, S.H.W., and Welch, J.G. 2000. Design parameters for post-ruminal drug delivery systems and rumen-stable products. In Controlled Release Veterinary Drug Delivery: Biological and Pharmaceutical Considerations. pp 83-113. Eds. Rathbone, M.J., and R. Gurny. Elsevier Scientific. Amsterdam.
- Fox, D.G., Tylutki, T.P., Van Amburgh, M.E., Chase, L.E., Pell, A.N., Overton, T.R., Tedeschi, L.O., Rasmussen, C.N., and Durbal V.M. 2000. The Net Carbohydrate and Protein System for Evaluating Herd Nutrition and Nutrient Excretion, CNCPS Version 4.0, Animal Science Mimeo 213, Cornell University, Ithaca, NY.
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