Boye, Mette

Jensen, Tim Kåre

Schou, Kirstine Klitgaard

Weiss Nielsen , Martin

Objective: To develop a fast and efficient method for measuring prevalence and gene expression of representatives from the three major groups of Treponema, most frequently identified in DD biopsies from cattle

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Downloaded from orbit.dtu.dk on: Dec 20, 2017High throughput quantitative PCR to measure prevalence and gene expression of thethree major groups of treponemes associated with Digital dermatitis in dairy cows.Schou, Kirstine Klitgaard; Weiss Nielsen , Martin ; Jensen, Tim Kåre; Boye, MettePublication date:2014Link back to DTU OrbitCitation (APA):Schou, K. K., Weiss Nielsen , M., Jensen, T. K., & Boye, M. (2014). High throughput quantitative PCR tomeasure prevalence and gene expression of the three major groups of treponemes associated with Digitaldermatitis in dairy cows.. Poster session presented at Gordon Research Conferences - Unique Features ofSpirochete-Host-Environment Interactions , Ventura, United States.High throughput quantitative PCR to measure prevalence and gene expression of the three major groups of treponemes associated with Digital dermatitis in dairy cows Kirstine Klitgaard, Martin W Nielsen, Tim K. Jensen & Mette Boye Objective  To develop a fast and efficient method for measuring prevalence and gene expression of representatives from the three major groups of Treponema, most frequently identified in DD biopsies from cattle.   References  1.  Ellen RP. 2006. Virulence Determinants of Oral Treponemes. In: Radolf JD and Lukehart SA (eds). Pathogenic Treponema. Molecular and Cellular Biology. Caister Academic Press, Norfolk, England, pp 357-386.  2. Evans NJ, Brown JM, Demirkan I, Murray RD, Vink WD, Blowey RW, Hart CA and Carter SD . 2008. Three unique groups of spirochetes isolated from digital dermatitis lesions in UK cattle. Vet Microbiol 130:141 -150.  3. Klitgaard K, Foix BA, Boye M and Jensen TK. 2013. Targeting the treponemal microbiome of digital dermatitis infections by high-resolution phylogenetic analyses and comparison with fluorescent in situ hybridization. J Clin Microbiol 51: 2212-2219.  4. Nordhoff M, Moter A, Schrank K and Wieler LH . 2008. High prevalence of treponemes in bovine digital dermatitis-A molecular epidemiology. Vet Microbiol. 131: 293 -300.  Introduction  Bovine digital dermatitis (DD), which causes lameness in cattle (Figure 1), is the most serious foot disorder of dairy cows from economic and welfare perspectives. Recent research point towards a polytreponemal etiology of infections, with Treponema phagedenis-like, Treponema medium/Treponema vincentii-like, and Treponema denticola/Treponema pedis-like phylotypes being highly associated with the disease2,3,4. Still the complex interplay between infecting Treponema species and their relative contributions to pathogenesis is largely unknown for this disease and must be elucidated.  Results   Although all three major groups of DD-related treponemes appeared to be present in most of the lesions (16S rRNA positive results), the expression results indicated that only T. phagedenis-like species were metabolically active (Figure 2). T. denticola/T. pedis-like and T. medium/T. vincentii-like species may either be present in low numbers and/or they may be less active than T. phagedenis. An example of the distribution of T. phagedenis-like species in DD lesions is shown in Figure 1b. From this picture it can be seen that T. phagedenis–like bacteria are highly active in the deeper part of the lesions, which is in good accordance with the expression results. The most active genes were related to functions such as host adhesion and motility (Figure 3). Figure 2. Heat map of expression analysis on 48.48 Dynamic array.  Cq values Target genes Sample no. Methods  From NCBI sequence data (http://www.ncbi.nlm.nih.gov/) we designed a high-throughput quantitative PCR (qPCR) assay to differentiate between T. denticola-like, T. medium/vincentii-like, and T. phagedenis-like phylotypes. Samples: cDNA from biopsies taken from cows just after slaughter: Controls (n=3), different stages of DD lesions (n=25). High-throughput reverse transcription qPCR analysis was performed on a  48.48 Dynamic Array (Fluidigm) (Figure 2), targeting 9 putative virulence genes1 and 3 reference genes (Table 1). 16S rRNA genes were applied for species identification. Table 1. Design of 48.48 Dynamic array.  a) Figure 1. Digital dermatitis lesion (DD12) at a proliferative stage: a) macroscopic, b) microscopic depiction of the same lesion. Bacteria are visualized by Fluorescence in situ hybridization applying a T. phagedenis specific probe (red) and a general eubacterial probe (green).  b) Gene name Function Putative virulence genes   Msp Major outer sheath protein. Binding to fibronectin, laminin, collagen types I and IV, hyaluronic acid. Hly Hemolycin. Exhibits hemooxidative and hemolytic activities FlgG Flagellar basal body rod protein FlgE Flagellar hook protein hbpB Hemin-binding protein B Lipoprotein PrtP  Serine protease, dentilisin Possibly involved in bacterial adhesion, tissue penetration and immune evasion  flaA Flagellar filament outer layer protein licCA Cholinephosphate cytidylyltransferase  mglA Galactose/methyl galactoside import ATP-binding protein Reference genes   hexo Hexokinase family protein (glycolysis) tpiA Triosephosphate isomerase (Glycolysis) lnt Apolipoprotein N-acyltransferase (Lipid metabolism) 16S rRNA Specific for T. pedis, T. phagedenis, T. medium/vincentii and T. denticola  Conclusion  The method of parallel gene expression analysis presented here needs further optimization but seems promising and may be broadly applicable for high throughput investigation of the infection dynamics of Treponema in DD lesions.  Figure 3. Mean expression (log2) of T. phagedenis-like phylotypes in positive samples (n=23).  024681012msp hly flgG flgE flaA hbpB prtP licCA mglA yeaZng (log2) Acknowledgements: Anastasia Isbrand is acknowledged for her technical support in the laboratory.  Funding: The project is funded by The Danish Council for Independent Research (project no. 12-127537).   

High throughput quantitative PCR to measure prevalence and gene expression of the three major groups of treponemes associated with Digital dermatitis in dairy cows.

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High throughput quantitative PCR to measure prevalence and gene expression of the three major groups of treponemes associated with Digital dermatitis in dairy cows.

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