Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP)

Scot E Dowd, Todd R Callaway, Randall D Wolcott, Yan Sun, Trevor McKeehan, Robert G Hagevoort, Thomas S Edrington, Scot E Dowd, Todd R Callaway, Randall D Wolcott, Yan Sun, Trevor McKeehan, Robert G Hagevoort, Thomas S Edrington

Abstract

Background: The microbiota of an animal's intestinal tract plays important roles in the animal's overall health, productivity and well-being. There is still a scarcity of information on the microbial diversity in the gut of livestock species such as cattle. The primary reason for this lack of data relates to the expense of methods needed to generate such data. Here we have utilized a bacterial tag-encoded FLX 16s rDNA amplicon pyrosequencing (bTEFAP) approach that is able to perform diversity analyses of gastrointestinal populations. bTEFAP is relatively inexpensive in terms of both time and labor due to the implementation of a novel tag priming method and an efficient bioinformatics pipeline. We have evaluated the microbiome from the feces of 20 commercial, lactating dairy cows.

Results: Ubiquitous bacteria detected from the cattle feces included Clostridium, Bacteroides, Porpyhyromonas, Ruminococcus, Alistipes, Lachnospiraceae, Prevotella, Lachnospira, Enterococcus, Oscillospira, Cytophage, Anaerotruncus, and Acidaminococcus spp. Foodborne pathogenic bacteria were detected in several of the cattle, a total of 4 cows were found to be positive for Salmonella spp (tentative enterica) and 6 cows were positive for Campylobacter spp. (tentative lanienae).

Conclusion: Using bTEFAP we have examined the microbiota in the feces of cattle. As these methods continue to mature we will better understand the ecology of the major populations of bacteria the lower intestinal tract. This in turn will allow for a better understanding of ways in which the intestinal microbiome contributes to animal health, productivity and wellbeing.

References

    1. Hungate RE. The Rumen and its microbes. New York, NY, Academic Press; 1966.
    1. Yokoyama MG, Johnson KA. Microbiology of the rumen and intestine. In: Church DC, editor. The Ruminant Animal: Digestive Physiology and Nutrition. Englewood Cliffs, N.J., Waveland Press; 1988. pp. 125–144.
    1. Dunkley KD, Dunkley CS, Njongmeta NL, Callaway TR, Hume ME, Kubena LF, Nisbet DJ, Ricke SC. Comparison of in vitro fermentation and molecular microbial profiles of high-fiber feed substrates incubated with chicken cecal inocula. Poult Sci. 2007;86:801–810.
    1. Callaway TR, Edrington TS, Anderson RC, Byrd JA, Nisbet DJ. Gastrointestinal microbial ecology and the safety of our food supply as related to Salmonella. J Anim Sci. 2007.
    1. Canibe N, Hojberg O, Hojsgaard S, Jensen BB. Feed physical form and formic acid addition to the feed affect the gastrointestinal ecology and growth performance of growing pigs. J Anim Sci. 2005;83:1287–1302.
    1. Flint HJ, Duncan SH, Scott KP, Louis P. Interactions and competition within the microbial community of the human colon: links between diet and health. Environ Microbiol. 2007;9:1101–1111.
    1. Flint HJ. Polysaccharide breakdown by anaerobic microorganisms inhabiting the Mammalian gut. Adv Appl Microbiol. 2004;56:89–120.
    1. Walker AW, Duncan SH, William Leitch EC, Child MW, Flint HJ. pH and peptide supply can radically alter bacterial populations and short-chain fatty acid ratios within microbial communities from the human colon. Appl Environ Microbiol. 2005;71:3692–3700.
    1. Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, Gill SR, Nelson KE, Relman DA. Diversity of the human intestinal microbial flora. Science. 2005;308:1635–1638.
    1. Ley RE, Peterson DA, Gordon JI. Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell. 2006;124:837–848.
    1. Pryde SE, Richardson AJ, Stewart CS, Flint HJ. Molecular analysis of the microbial diversity present in the colonic wall, colonic lumen, and cecal lumen of a pig. Appl Environ Microbiol. 1999;65:5372–5377.
    1. Zhu WY, Williams BA, Konstantinov SR, Tamminga S, de Vos WM, Akkermans AD. Analysis of 16S rDNA reveals bacterial shift during in vitro fermentation of fermentable carbohydrate using piglet faeces as inoculum. Anaerobe. 2003;9:175–180.
    1. Flint HJ. The rumen microbial ecosystem--some recent developments. Trends Microbiol. 1997;5:483–488.
    1. Jenny BF, Vandijk HJ, Collins JA. Performance and fecal flora of calves fed a Bacillus subtilis concentrate. J Dairy Sci. 1991;74:1968–1973.
    1. Rada V, Vlkova E, Nevoral J, Trojanova I. Comparison of bacterial flora and enzymatic activity in faeces of infants and calves. FEMS Microbiol Lett. 2006;258:25–28.
    1. Nocker A, Burr M, Camper AK. Genotypic microbial community profiling: a critical technical review. Microb Ecol. 2007;54:276–289.
    1. Dahllof I. Molecular community analysis of microbial diversity. Curr Opin Biotechnol. 2002;13:213–217.
    1. Farrelly V, Rainey FA, Stackebrandt E. Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species. Appl Environ Microbiol. 1995;61:2798–2801.
    1. von WF, Gobel UB, Stackebrandt E. Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev. 1997;21:213–229.
    1. Suzuki MT, Giovannoni SJ. Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl Environ Microbiol. 1996;62:625–630.
    1. Dowd SE, Sun Y, Secor PR, Rhoads DD, Wolcott BM, James GA, Wolcott RD. Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosome shotgun sequencing. BMC Microbiol. 2008;8:43.
    1. Roesch LF, Fulthorpe RR, Riva A, Casella G, Hadwin AK, Kent AD, Daroub SH, Camargo FA, Farmerie WG, Triplett EW. Pyrosequencing enumerates and contrasts soil microbial diversity. ISME J. 2007;1:283–290.
    1. Huse SM, Huber JA, Morrison HG, Sogin ML, Welch DM. Accuracy and quality of massively parallel DNA pyrosequencing. Genome Biol. 2007;8:R143.
    1. Cristea-Fernstrom M, Olofsson M, Chryssanthou E, Jonasson J, Petrini B. Pyrosequencing of a short hypervariable 16S rDNA fragment for the identification of nontuberculous mycobacteria--a comparison with conventional 16S rDNA sequencing and phenotyping. APMIS. 2007;115:1252–1259.
    1. Liu Z, Lozupone C, Hamady M, Bushman FD, Knight R. Short pyrosequencing reads suffice for accurate microbial community analysis. Nucleic Acids Res. 2007;35:e120.
    1. Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: human gut microbes associated with obesity. Nature. 2006;444:1022–1023.
    1. Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature. 2006;444:1027–1031.
    1. Bolte ER. Autism and Clostridium tetani. Med Hypotheses. 1998;51:133–144.
    1. Finegold SM, Molitoris D, Song Y, Liu C, Vaisanen ML, Bolte E, McTeague M, Sandler R, Wexler H, Marlowe EM, Collins MD, Lawson PA, Summanen P, Baysallar M, Tomzynski TJ, Read E, Johnson E, Rolfe R, Nasir P, Shah H, Haake DA, Manning P, Kaul A. Gastrointestinal microflora studies in late-onset autism. Clin Infect Dis. 2002;35:S6–S16.
    1. Vanbelle M, Teller E, Focant M. Probiotics in animal nutrition: a review. Arch Tierernahr. 1990;40:543–567.
    1. Ricke SC, Pillai SD. Conventional and molecular methods for understanding probiotic bacteria functionality in gastrointestinal tracts. Crit Rev Microbiol. 1999;25:19–38.
    1. Drasar BS, Barrow PA. Intestinal Microbiology. Wokingham, UK, Van Nostrand Reinhold; 1985.
    1. Grizard D, Barthomeuf C. Non-digestible oligosaccharides used as prebiotic agents: mode of production and beneficial effects on animal and human health. Reprod Nutr Dev. 1999;39:563–588.
    1. Kanauchi O, Matsumoto Y, Matsumura M, Fukuoka M, Bamba T. The beneficial effects of microflora, especially obligate anaerobes, and their products on the colonic environment in inflammatory bowel disease. Curr Pharm Des. 2005;11:1047–1053.
    1. Attwood G, Li D, Pacheco D, Tavendale M. Production of indolic compounds by rumen bacteria isolated from grazing ruminants. J Appl Microbiol. 2006;100:1261–1271.
    1. Songer JG. The emergence of Clostridium difficile as a pathogen of food animals. Anim Health Res Rev. 2004;5:321–326.
    1. Songer JG. Clostridial diseases of small ruminants. Vet Res. 1998;29:219–232.
    1. Reilly K, Attwood GT. Detection of Clostridium proteoclasticum and closely related strains in the rumen by competitive PCR. Appl Environ Microbiol. 1998;64:907–913.
    1. Widyastuti Y, Lee SK, Suzuki K, Mitsuoka T. Isolation and characterization of rice-straw degrading clostridia from cattle rumen. J Vet Med Sci. 1992;54:185–188.
    1. Kopecny J, Hodrova B, Stewart CS. The effect of rumen chitinolytic bacteria on cellulolytic anaerobic fungi. Lett Appl Microbiol. 1996;23:199–202.
    1. van der Wielen PW, Lipman LJ, van KF, Biesterveld S. Competitive exclusion of Salmonella enterica serovar Enteritidis by Lactobacillus crispatus and Clostridium lactatifermentans in a sequencing fed-batch culture. Appl Environ Microbiol. 2002;68:555–559.
    1. Leser TD, Amenuvor JZ, Jensen TK, Lindecrona RH, Boye M, Moller K. Culture-independent analysis of gut bacteria: the pig gastrointestinal tract microbiota revisited. Appl Environ Microbiol. 2002;68:673–690.
    1. Ozutsumi Y, Hayashi H, Sakamoto M, Itabashi H, Benno Y. Culture-independent analysis of fecal microbiota in cattle. Biosci Biotechnol Biochem. 2005;69:1793–1797.
    1. Wexler HM. Bacteroides: the good, the bad, and the nitty-gritty. Clin Microbiol Rev. 2007;20:593–621.
    1. Shoemaker NB, Anderson KL, Smithson SL, Wang GR, Salyers AA. Conjugal transfer of a shuttle vector from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4. Appl Environ Microbiol. 1991;57:2114–2120.
    1. Elad D, Friedgut O, Alpert N, Stram Y, Lahav D, Tiomkin D, Avramson M, Grinberg K, Bernstein M. Bovine necrotic vulvovaginitis associated with Porphyromonas levii. Emerg Infect Dis. 2004;10:505–507.
    1. Walter MR, Morck DW. In vitro expression of tumor necrosis factor-alpha, interleukin 1beta, and interleukin 8 mRNA by bovine macrophages following exposure to Porphyromonas levii. Can J Vet Res. 2002;66:93–98.
    1. Wise MG, Siragusa GR. Quantitative analysis of the intestinal bacterial community in one- to three-week-old commercially reared broiler chickens fed conventional or antibiotic-free vegetable-based diets. J Appl Microbiol. 2007;102:1138–1149.
    1. Avgustin G, Wallace RJ, Flint HJ. Phenotypic diversity among ruminal isolates of Prevotella ruminicola: proposal of Prevotella brevis sp. nov., Prevotella bryantii sp. nov., and Prevotella albensis sp. nov. and redefinition of Prevotella ruminicola. Int J Syst Bacteriol. 1997;47:284–288.
    1. Rautio M, Eerola E, Vaisanen-Tunkelrott ML, Molitoris D, Lawson P, Collins MD, Jousimies-Somer H. Reclassification of Bacteroides putredinis (Weinberg et al., 1937) in a new genus Alistipes gen. nov., as Alistipes putredinis comb. nov., and description of Alistipes finegoldii sp. nov., from human sources. Syst Appl Microbiol. 2003;26:182–188.
    1. Scupham J, Patton TG, Bent E, Bayles DO. Comparison of the Cecal Microbiota of Domestic and Wild Turkeys. Microb Ecol. 2008.
    1. Hernandez JD, Scott PT, Shephard RW, Al Jassim RA. The characterization of lactic acid producing bacteria from the rumen of dairy cattle grazing on improved pasture supplemented with wheat and barley grain. J Appl Microbiol. 2008
    1. Uyeno Y, Sekiguchi Y, Tajima K, Takenaka A, Kurihara M, Kamagata Y. Evaluation of group-specific, 16S rRNA-targeted scissor probes for quantitative detection of predominant bacterial populations in dairy cattle rumen. J Appl Microbiol. 2007;103:1995–2005.
    1. Min BR, Pinchak WE, Anderson RC, Hume ME. In vitro bacterial growth and in vivo ruminal microbiota populations associated with bloat in steers grazing wheat forage. J Anim Sci. 2006;84:2873–2882.
    1. Cornick NA, Jensen NS, Stahl DA, Hartman PA, Allison MJ. Lachnospira pectinoschiza sp. nov., an anaerobic pectinophile from the pig intestine. Int J Syst Bacteriol. 1994;44:87–93.
    1. Harmsen HJ, Raangs GC, He T, Degener JE, Welling GW. Extensive set of 16S rRNA-based probes for detection of bacteria in human feces. Appl Environ Microbiol. 2002;68:2982–2990.
    1. Jay MT, Cooley M, Carychao D, Wiscomb GW, Sweitzer RA, Crawford-Miksza L, Farrar JA, Lau DK, O'Connell J, Millington A, Asmundson RV, Atwill ER, Mandrell RE. Escherichia coli O157:H7 in feral swine near spinach fields and cattle, central California coast. Emerg Infect Dis. 2007;13:1908–1911.
    1. Shelton DR, Karns JS, Higgins JA, Van Kessel JA, Perdue ML, Belt KT, Russell-Anelli J, Debroy C. Impact of microbial diversity on rapid detection of enterohemorrhagic Escherichia coli in surface waters. FEMS Microbiol Lett. 2006;261:95–101.
    1. Rodriguez A, Pangloli P, Richards HA, Mount JR, Draughon FA. Prevalence of Salmonella in diverse environmental farm samples. J Food Prot. 2006;69:2576–2580.
    1. Vanselow BA, Hum S, Hornitzky MA, Eamens GJ, Quinn K. Salmonella Typhimurium persistence in a Hunter Valley dairy herd. Aust Vet J. 2007;85:446–450.
    1. Callaway TR, Anderson RC, Edrington TS, Genovese KJ, Harvey RB, Poole TL, Nisbet DJ. Recent pre-harvest supplementation strategies to reduce carriage and shedding of zoonotic enteric bacterial pathogens in food animals. Anim Health Res Rev. 2004;5:35–47.
    1. Callaway TR, Dunkley KD, Anderson RC, Edrington TS, Genovese KJ, Poole TL, Nisbet DJ. Probiotics, vaccines and other intervention strategies. In: Sofos JN, editor. Raw Material Safety: Meat. Cabridge, UK., Woohead Pub.; 2004. pp. 192–213.
    1. Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, Griffin PM, Tauxe RV. Food-related illness and death in the United States. Emerg Infect Dis. 1999;5:607–625.
    1. Centers for Disease Control and Prevention Multistate outbreak of Salmonella typhimurium infections associated with eating ground beef--United States, 2004. MMWR Morb Mortal Wkly Rep. 2006;55:180–182.
    1. Centers for Disease Control and Prevention Outbreak of multidrug-resistant Salmonella newport--United States, January-April 2002. MMWR Morb Mortal Wkly Rep. 2002;51:545–548.
    1. Dechet AM, Scallan E, Gensheimer K, Hoekstra R, Gunderman-King J, Lockett J, Wrigley D, Chege W, Sobel J. Outbreak of multidrug-resistant Salmonellaenterica serotype Typhimurium Definitive Type 104 infection linked to commercial ground beef, northeastern United States, 2003-2004. Clin Infect Dis. 2006;42:747–752.
    1. Naylor SW, Low JC, Besser TE, Mahajan A, Gunn GJ, Pearce MC, McKendrick IJ, Smith DG, Gally DL. Lymphoid follicle-dense mucosa at the terminal rectum is the principal site of colonization of enterohemorrhagic Escherichia coli O157:H7 in the bovine host. Infect Immun. 2003;71:1505–1512.
    1. Choi JH, Lee SH, Fukushi K, Yamamoto K. Comparison of sludge characteristics and PCR-DGGE based microbial diversity of nanofiltration and microfiltration membrane bioreactors. Chemosphere. 2007;67:1543–1550.
    1. Binder A, Amtsberg G, Stock V, Bisping W. [Occurrence of Gram-negative anaerobic bacteria and clostridia in fecal flora of clinically healthy swine and weaned piglets with swine dysentery and nutritional diarrhea] Zentralbl Veterinarmed B. 1984;31:401–412.
    1. Wittenbrink MM, Amtsberg G, Kamphues J. [Intestinal and fecal flora of weaned piglets with nutritive diarrhea caused by forced feed intake] Dtsch Tierarztl Wochenschr. 1984;91:387–391.
    1. Bryant MP. Normal flora--rumen bacteria. Am J Clin Nutr. 1970;23:1440–1450.
    1. Attebery HR, Sutter VL, Finegold SM. Effect of a partially chemically defined diet on normal human fecal flora. Am J Clin Nutr. 1972;25:1391–1398.
    1. Biesheuvel MH, Bijker PG, Urlings HA. Some aspects of the gastrointestinal microflora of veal calves fed different rations: a pilot study. Vet Q. 1991;13:97–104.
    1. Ellinger DK, Muller LD, Glantz PJ. Influence of feeding fermented colostrum and Lactobacillus acidophilus on fecal flora of dairy calves. J Dairy Sci. 1980;63:478–482.
    1. Hara H, Orita N, Hatano S, Ichikawa H, Hara Y, Matsumoto N, Kimura Y, Terada A, Mitsuoka T. Effect of tea polyphenols on fecal flora and fecal metabolic products of pigs. J Vet Med Sci. 1995;57:45–49.
    1. Huang X, Madan A. CAP3: A DNA sequence assembly program. Genome Res. 1999;9:868–877.
    1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–410.
    1. Maidak BL, Cole JR, Lilburn TG, Parker CT, Jr., Saxman PR, Farris RJ, Garrity GM, Olsen GJ, Schmidt TM, Tiedje JM. The RDP-II (Ribosomal Database Project) Nucleic Acids Res. 2001;29:173–174.

Source: PubMed

3
Subscribe