Current Microbiology

, Volume 58, Issue 4, pp 294–299 | Cite as

Use of Real-Time PCR Technique in Studying Rumen Cellulolytic Bacteria Population as Affected by Level of Roughage in Swamp Buffalo



A real-time polymerase chain reaction approach was used in this study to determine the population of major ruminal bacterial species (Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) in digesta and rumen fluid of swamp buffalo (Bubalus bubalis). Four rumen-fistulated, male swamp buffalo were randomly assigned according to a 4 × 4 Latin square design to evaluate the effect of the urea-treated rice straw (roughage source)-to-concentrate ratio on cellulolytic bacterial distribution. Animals were fed roughage-to-concentrate (R:C) ratios of 100:0, 75:25, 50:50, and 25:75, respectively. At the end of each period, rumen fluid and digesta were collected at 0 h and 4 h post-morning-feeding. It was found that feeding urea-treated rice straw solely increased these three cellulolytic bacteria numbers up to 2.65 × 109 and 3.54 × 109 copies per milliliter for F. succinogenes, 5.10 × 107 and 7.40 × 107 copies per millilter for R. Flavefaciens, and 4.00 × 106 and 6.00 × 106 copies per milliliter for R. albus in rumen fluid and digesta, respectively. The distribution of the three cellulolytic bacteria species in digesta were highest at 3.21 × 109, 4.55 × 107, and 4.56 × 106 copies per milliliter for F. succinogenes, R. flavefaciens, and R. albus, respectively. Moreover, at 4 h post-morning-feeding, the populations of the three cellulolytic bacteria were higher than found at 0 h post-morning-feeding. It is most notable that F. succinogenes were the highest in population in the rumen of swamp buffalo and cellulolytic bacteria mostly adhered to feed digesta in the rumen.


  1. 1.
    Briesacher SL, May T, Grigsby KN, Kerley MS, Anthony RV, Paterson JA (1992) Use of DNA probes to monitor nutritional effects on ruminal prokaryotes and Fibrobacter succinogenes S85. J Anim Sci 70:289–295PubMedGoogle Scholar
  2. 2.
    Bryant MP (1959) Bacterial species of the rumen. Bacteriol Rev 23:125–153PubMedGoogle Scholar
  3. 3.
    Castillo M, Martin-Orue MS, Manzanilla EG, Badiola I, Gasa MJ (2006) Quantification of total bacteria, enterobacteria and lactobacilli populations in pig digesta by real-time PCR. Vet Microbiol 114:165–170PubMedCrossRefGoogle Scholar
  4. 4.
    Collado MC, Sanz Y (2006) Quantification of mucosa-adhered microbiota of lambs and calves by the use of culture methods and fluorescent in situ hybridization coupled with flow cytometry techniques. Vet Microbiol 121:299–306PubMedCrossRefGoogle Scholar
  5. 5.
    Denman SE, McSweeney CS (2006) Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen. FEMS Microbiol Ecol 58:572–582PubMedCrossRefGoogle Scholar
  6. 6.
    Forster RJ, Gong J, Teather RM (1997) Group-specific 16S rRNA hybridization probes for determinative and community structure studies of Butyrivibrio fibrisolvens in the rumen. Appl Environ Microbiol 63:1256–1260PubMedGoogle Scholar
  7. 7.
    Hungate RE (1966) The rumen and its microbes. Academic Press, New York, pp 53Google Scholar
  8. 8.
    Kobayashi Y (2006) Inclusion of novel bacteria in rumen microbiology: need for basic and applied science. Anim Sci J 77:375–385CrossRefGoogle Scholar
  9. 9.
    Koike S, Kobayashi Y (2001) Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. FEMS Microbiol Lett 204:361–366PubMedCrossRefGoogle Scholar
  10. 10.
    Koike S, Pan J, Kobayashi Y, Tanaka K (2003) Kinetics of in sacco fiber-attachment of representative ruminal cellulolytic bacteria monitored by competitive PCR. J Dairy Sci 86:1429–1435PubMedGoogle Scholar
  11. 11.
    Krause DO, Dalrymple BP, Smith WJ, Mackie RI, McSweeney CS (1999) 16S rDNA sequencing of Ruminococcus albus and Ruminococcus flavefaciens: design of a signature probe and its application in adult sheep. Microbiology 145:1797–1807PubMedCrossRefGoogle Scholar
  12. 12.
    Leser TD, Boye M, Hendriksen NB (1995) Survival and activity of a Pseudomonas sp. strain B13 (FR1) in a marine microcosm determined by quantitative PCR and an rRNA-targeting probe and its effect on the indigenous bacterioplankton. Appl Environ Microbiol 61:1201–1207PubMedGoogle Scholar
  13. 13.
    McSweeney CS, Denman E (2007) Effect of sulfur supplements on cellulolytic rumen micro-organisms and microbial protein synthesis in cattle fed a high fibre diet. Appl Microbiol 103:1757–1765CrossRefGoogle Scholar
  14. 14.
    Miron J, Ben-Ghedalia D, Morrison M (2001) Invited review: adhesion mechanisms of rumen cellulolytic bacteria. J Dairy Sci 84:1294–1309PubMedCrossRefGoogle Scholar
  15. 15.
    Mosoni P, Durand FC, Maillet CB, Forano E (2007) Quantification by real-time PCR of cellulolytic bacteria in the rumen of sheep after supplementation of a forage diet with readily fermentable carbohydrates: effect of a yeast additive. Appl Microbiol 103:2676–2685CrossRefGoogle Scholar
  16. 16.
    Qi M, Nelson KE, Daugherty SC et al (2008) Genomic differences between Fibrobacter succinogenes S85 and Fibrobacter intestinalis DR7, identified by suppression subtractive hybridization. Appl Environ Microbiol 74:987–993PubMedCrossRefGoogle Scholar
  17. 17.
    Reilly K, Attwood GT (1998) Detection of Clostridium proteoclasticum and closely related strains in the rumen by competitive PCR. Appl Environ Microbiol 64:907–913PubMedGoogle Scholar
  18. 18.
    Russell JB, Rychlik JL (2001) Factors that alter rumen microbial ecology. Science 292:1119–1122PubMedCrossRefGoogle Scholar
  19. 19.
    AS S (1998) User’s guide: Statistics, Version, vol 7. SAS Institute, Inc., Cary, NCGoogle Scholar
  20. 20.
    Shinkai T, Kobayashi Y (2007) Localization of ruminal cellulolytic bacteria on plant fibrous materials as determined by fluorescence in situ hybridization and real-time PCR. Appl Environ Microbiol 73:1646–1652PubMedCrossRefGoogle Scholar
  21. 21.
    Skillman LC, Toovey AF, Williams AJ, Wright AD (2006) Development and validation of a real-time PCR method to quantify rumen protozoa and examination of variability between entodinium populations in sheep offered a hay-based diet. Appl Environ Microbial 72:200–206CrossRefGoogle Scholar
  22. 22.
    Stahl DA, Flesher B, Mansfield HR, Montgomery L (1988) Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Appl Environ Microbiol 54:1079–1084PubMedGoogle Scholar
  23. 23.
    Steel RGD, Torrie JH (1980) Principles and procedure of statistics. McGraw Hill, New YorkGoogle Scholar
  24. 24.
    Sung HG, Kobayashi Y, Chang J, Ha A, Hwang H, Ha JK (2007) Low ruminal pH reduces dietary fiber digestion via reduced microbial attachment. Asian-Aust J Anim Sci 20:200–207Google Scholar
  25. 25.
    Sylvester JT, Karnati SK, Yu Z, Morrison M, Firkins JL (2004) Development of an assay to quantify rumen ciliate protozoal biomass in cows using real-time PCR. J Nutr 134:3378–3384PubMedGoogle Scholar
  26. 26.
    Tajima K, Nagamine T, Matsui H, Nakamura M, Aminov RI (2001) Phylogenetic analysis of archaeal 16S rRNA libraries from the rumen suggests the existence of a novel group of archaea not associated with known methanogens. FEMS Microbiol Lett 200:67–72PubMedCrossRefGoogle Scholar
  27. 27.
    Wanapat M (1985) Improving rice straw quality as ruminant feed by urea-treated in Thailand. In: Wanapat M, Devendra C (eds) Proceedings of relevance of crops residues as animal feeds in developing countries. Bangkok, Thailand, pp 147–175Google Scholar
  28. 28.
    Wanapat M (2000) Rumen manipulation to increase the efficient use local feed resources and productivity of ruminants in the tropics. In: Stone GM (ed) Proceedings of the 9th AAAP Congress. University of New South Wales, Sydney, Australia, pp 59–67Google Scholar
  29. 29.
    Wanapat M, Rowlinson P (2007) Nutrition and feeding of swamp buffalo: feed resources and rumen approach. In: Palo RD (ed), Proceedings of the 8th World Buffalo Congress. Caserta, Italy, pp 67–73Google Scholar
  30. 30.
    Wora-anu S, Wanapat M, Wachirapakorn C, Nuntaso N (2000) Effects of roughage to concentrate ratio on ruminal ecology and voluntary feed intake in cattle and swamp buffaloes fed on urea-treated rice straw. In: Stone GM (ed), Proceedings of the 9th AAAP Congress. University of New South Wales, Sydney, Australia, p 236 (supplement)Google Scholar
  31. 31.
    Yu Z, Morrison M (2004) Improved extraction of PCR-quality community DNA from digesta and fecal samples. BioTechniques 36:808–812PubMedGoogle Scholar
  32. 32.
    Zimmermann K, Mannhalter JW (1996) Technical aspects of quantitative competitive PCR. BioTechniques 21:268–279PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  1. 1.Department of Animal Science, Faculty of Agriculture, Tropical Feed Resources Research and Development Center (TROFREC)Khon Kaen UniversityKhon KaenThailand

Personalised recommendations