All Title Author
Keywords Abstract

PLOS ONE  2012 

Microbial Diversity of Bovine Mastitic Milk as Described by Pyrosequencing of Metagenomic 16s rDNA

DOI: 10.1371/journal.pone.0047671

Full-Text   Cite this paper   Add to My Lib


Dairy cow mastitis is an important disease in the dairy industry. Different microbial species have been identified as causative agents in mastitis, and are traditionally diagnosed by bacterial culture. The objective of this study was to use metagenomic pyrosequencing of bacterial 16S rRNA genes to investigate bacterial DNA diversity in milk samples of mastitic and healthy dairy cows and compare the results with those obtained by classical bacterial culture. One hundred and thirty-six milk samples were collected from cows showing signs of mastitis and used for microbiological culture. Additionally, 20 milk samples were collected from healthy quarters. Bacterial DNA was isolated from the same milk samples and the 16S rRNA genes were individually amplified and pyrosequenced. Discriminant analysis showed that the groups of samples that were most clearly different from the rest and thus easily discriminated were the normal milk samples from healthy cows and those characterised by culture as Trueperella pyogenes and Streptococcus spp. The mastitis pathogens identified by culture were generally among the most frequent organisms detected by pyrosequencing, and in some cases (Escherichia coli, Klebsiella spp. and Streptococcus uberis mastitis) the single most prevalent microorganism. Trueperella pyogenes sequences were the second most prevalent sequences in mastitis cases diagnosed as Trueperella pyogenes by culture, Streptococcus dysgalactiae sequences were the second most prevalent sequences in mastitis cases diagnosed as Streptococcus dysgalactiae by culture, and Staphyloccocus aureus sequences were the third most prevalent in mastitis cases diagnosed as Staphylococcus aureus by culture. In samples that were aerobic culture negative, pyrosequencing identified DNA of bacteria that are known to cause mastitis, DNA of bacteria that are known pathogens but have so far not been associated with mastitis, and DNA of bacteria that are currently not known to be pathogens. A possible role of anaerobic pathogens in bovine mastitis is also suggested.


[1]  Halasa T, Huijps K, Osteras O, Hogeveen H (2007) Economic effects of bovine mastitis and mastitis management: A review. Vet Q 29(1): 18–31.
[2]  Taponen S, Salmikivi L, Simojoki H, Koskinen MT, Pyorala S (2009) Real-time polymerase chain reaction-based identification of bacteria in milk samples from bovine clinical mastitis with no growth in conventional culturing. J Dairy Sci 92(6): 2610–2617.
[3]  Koskinen MT, Wellenberg GJ, Sampimon OC, Holopainen J, Rothkamp A, et al. (2010) Field comparison of real-time polymerase chain reaction and bacterial culture for identification of bovine mastitis bacteria. J Dairy Sci 93(12): 5707–5715.
[4]  Shome BR, Das Mitra S, Bhuvana M, Krithiga N, Velu D, et al. (2011) Multiplex PCR assay for species identification of bovine mastitis pathogens. J Appl Microbiol 111(6): 1349–1356.
[5]  Kuang Y, Tani K, Synnott AJ, Ohshima K, Higuchi H, et al. (2009) Characterization of bacterial population of raw milk from bovine mastitis by culture-independent PCR–DGGE method. Biochem Eng J 45(1): 76–81.
[6]  Schwaiger K, Wimmer M, Huber-Schlenstedt R, Fehlings K, Holzel CS, et al. (2012) Hot topic: Bovine milk samples yielding negative or nonspecific results in bacterial culturing–the possible role of PCR-single strand conformation polymorphism in mastitis diagnosis. J Dairy Sci 95(1): 98–101.
[7]  Hunt KM, Foster JA, Forney LJ, Schutte UM, Beck DL, et al. (2011) Characterization of the diversity and temporal stability of bacterial communities in human milk. PLoS One 6(6): e21313.
[8]  Kolbert CP, Persing DH (1999) Ribosomal DNA sequencing as a tool for identification of bacterial pathogens. Curr Opin Microbiol 2(3): 299–305.
[9]  Bhatt VD, Ahir VB, Koringa PG, Jakhesara SJ, Rank DN, et al. (2012) Milk microbiome signatures of subclinical mastitis-affected cattle analysed by shotgun sequencing. J Appl Microbiol 112(4): 639–650.
[10]  Sun Y, Cai Y, Mai V, Farmerie W, Yu F, et al. (2010) Advanced computational algorithms for microbial community analysis using massive 16S rRNA sequence data. Nucleic Acids Res 38(22): e205.
[11]  Py?r?l? S, Jousimies-Somer H, Mero M (1992) Clinical, bacteriological and therapeutic aspects of bovine mastitis caused by aerobic and anaerobic pathogens. Br Vet J 148(1): 54–62.
[12]  Elad D, Friedgut O, Alpert N, Stram Y, Lahav D, et al. (2004) Bovine necrotic vulvovaginitis associated with Porphyromonas levii. Emerg Infect Dis 10(3): 505–507.
[13]  Santos TM, Gilbert RO, Bicalho RC (2011) Metagenomic analysis of the uterine bacterial microbiota in healthy and metritic postpartum dairy cows. J Dairy Sci 94(1): 291–302.
[14]  Bicalho ML, Machado VS, Oikonomou G, Gilbert RO, Bicalho RC (2011) Association between virulence factors of Escherichia coli, Fusobacterium necrophorum, and Arcanobacterium pyogenes and uterine diseases of dairy cows. Vet Microbiol.
[15]  Nagaraja TG, Narayanan SK, Stewart GC, Chengappa MM (2005) Fusobacterium necrophorum infections in animals: Pathogenesis and pathogenic mechanisms. Anaerobe 11(4): 239–246.
[16]  Bexiga R, Koskinen MT, Holopainen J, Carneiro C, Pereira H, et al. (2011) Diagnosis of intramammary infection in samples yielding negative results or minor pathogens in conventional bacterial culturing. J Dairy Res 78(1): 49–55.
[17]  Poindexter JS (2009) Caulobacter. In: Editor-in-Chief:Moselio Schaechter, editor. Encyclopedia of Microbiology (Third Edition). Oxford: Academic Press. 57–73.
[18]  Tsakalidou E, Zoidou E, Pot B, Wassill L, Ludwig W, et al. (1998) Identification of Streptococci from greek kasseri cheese and description of Streptococcus macedonicus sp. nov. Int J Syst Bacteriol 48 Pt 2: 519–527.
[19]  Herrero IA, Rouse MS, Piper KE, Alyaseen SA, Steckelberg JM, et al. (2002) Reevaluation of Streptococcus bovis endocarditis cases from 1975 to 1985 by 16S ribosomal DNA sequence analysis. J Clin Microbiol 40(10): 3848–3850.
[20]  Luthje P, Schwarz S (2006) Antimicrobial resistance of coagulase-negative staphylococci from bovine subclinical mastitis with particular reference to macrolide-lincosamide resistance phenotypes and genotypes. J Antimicrob Chemother 57(5): 966–969.
[21]  Fessler AT, Billerbeck C, Kadlec K, Schwarz S (2010) Identification and characterization of methicillin-resistant coagulase-negative staphylococci from bovine mastitis. J Antimicrob Chemother 65(8): 1576–1582.
[22]  Pyorala S, Taponen S (2009) Coagulase-negative staphylococci-emerging mastitis pathogens. Vet Microbiol 134(1–2): 3–8.
[23]  Schukken YH, Gonzalez RN, Tikofsky LL, Schulte HF, Santisteban CG, et al. (2009) CNS mastitis: Nothing to worry about? Vet Microbiol 134(1–2): 9–14.
[24]  Kutzer P, Schulze C, Engelhardt A, Wieler LH, Nordhoff M (2008) Helcococcus ovis, an emerging pathogen in bovine valvular endocarditis. J Clin Microbiol 46(10): 3291–3295.
[25]  Collins MD, Falsen E, Foster G, Monasterio LR, Dominguez L, et al. (1999) Helcococcus ovis sp. nov., a gram-positive organism from sheep. Int J Syst Bacteriol 49 Pt 4: 1429–1432.
[26]  Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132: 365–386.
[27]  Drummond GB, Vowler SL (2012) Analysis of variance: Variably complex. J Physiol 590(Pt 6): 1303–1306.
[28]  Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73(16): 5261–5267.
[29]  Cole JR, Wang Q, Cardenas E, Fish J, Chai B, et al.. (2009) The ribosomal database project: Improved alignments and new tools for rRNA analysis. Nucleic Acids Res 37(Database issue): D141–5.
[30]  Nawrocki EP, Eddy SR (2007) Query-dependent banding (QDB) for faster RNA similarity searches. PLoS Comput Biol 3(3): e56.
[31]  Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3): 403–410.
[32]  Saitou N, Nei M (1987) The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4): 406–425.
[33]  Cantor CR, Jukes TH (1966) The repetition of homologous sequences in the polypetide chains of certain cytochromes and globins. Proc Natl Acad Sci U S A 56(1): 177–184.


comments powered by Disqus

Contact Us


微信:OALib Journal