Quantitative Detection of Clostridium perfringens in Broiler Chickens by Real-Time PCR Targeting the Alpha-Toxin Gene

Publikation: Bidrag til bog/antologi/rapport/proceedingKonferenceabstrakt i proceedingsForskning

  • Lone Abildgaard, Danmark
  • Ricarda M. Engberg, Danish Institute of Agricultural Sciences, Department of Animal Health, Welfare and Nutrition, Tjele, Danmark
  • Andreas Schramm
  • Ole Højberg, Danish Institute of Agricultural Sciences, Department of Animal Health, Welfare and Nutrition, Tjele, Danmark
  • Mikrobiologi, Biologisk Institut
QUANTITATIVE DETECTION OF CLOSTRIDIUM PERFRINGENS IN BROILER CHICKENS BY REAL-TIME PCR TARGETING THE ALPHA-TOXIN GENE

L. Abildgaard 1, R.M. Engberg 1, A. Schramm 2, O. Højberg 1
1 Danish Institute of Agricultural Sciences, Department of Animal Health, Welfare and Nutrition, Tjele, Denmark; 2 University of Aarhus, Institute of Biological Sciences, Department of Microbiology, Aarhus, Denmark

Necrotic enteritis is a severe gastrointestinal disease in broiler chickens caused by C. perfringens producing α-toxin (phospholipase C). The incidence of necrotic enteritis in broilers has been reduced by antibiotics (ionophores) presently used to prevent parasitic coccidiosis. From 2012 the European Union has banned these anticoccidials as feed additives, wherefore alternatives are needed to suppress C. perfringens and/or α-toxin production.

A real-time PCR primer-probe set targeting the α-toxin gene was developed by sequencing the α-toxin gene from ~60 strains of C. perfringens, isolated from diseased as well as healthy broilers. For its application to the chicken gastrointestinal tract (i.e., ileum), DNA extraction efficiency and potential inhibition of the real-time PCR process by ileum content was evaluated by (i) analysing ileum samples spiked with C. perfringens cells, and (ii) comparing DNA extractions/real-time PCR of pure cultures with and without ileum content added. No inhibition was observed, and the quantitative real-time PCR approach correlated well with counts in pure cultures and spiked ileum content. The protocol had a detection limit of ~1000 cells spiked into 200 mg ileal material. This matched the sensitivity obtained using published primers targeting C. perfringens 16S rDNA. However, targeting the functional α-toxin gene offers the possibility to assess not only abundance of C. perfringens but also the expression level (mRNA) of the virulence factor. The protocol is currently used in ileum samples from chickens fed with additives previously shown to suppress growth of C. perfringens in vitro; adaptation of the protocol to mRNA analysis is under way.
OriginalsprogEngelsk
TitelThe Hidden Powers - Microbial Communities in Action. Proceedings of the 11th International Symposium on Microbial Ecology (ISME-11)
Antal sider1
ForlagInternational Society for Microbial Ecology
Udgivelsesår2006
Sider441
StatusUdgivet - 2006
Begivenhed11th International Symposium on Microbial Ecology (ISME-11) - Vienna, Østrig
Varighed: 20 aug. 200625 aug. 2006

Konference

Konference11th International Symposium on Microbial Ecology (ISME-11)
LandØstrig
ByVienna
Periode20/08/200625/08/2006

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