Related Experiment Video
Updated: Jun 13, 2026
A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence
Published on: October 11, 2018
Quantitative real-time PCR assay for Clostridium septicum in poultry gangrenous dermatitis associated samples
A P Neumann1, S M Dunham, T G Rehberger
1Danisco USA Inc., Waukesha, WI 53186, USA. anthony.neumann@danisco.com
Abstract:
Clostridium septicum is a spore-forming anaerobe frequently implicated in cases of gangrenous dermatitis (GD) and other spontaneously occurring myonecrotic infections of poultry. Although C. septicum is readily cultured from diseased tissues it can be difficult to enumerate due to its tendency to swarm over the surface of agar plates. In this study a quantitative real-time PCR assay was developed in order to more accurately measure the levels of C. septicum in healthy as well as GD associated poultry samples. The assay was specifically designed to target the C. septicum alpha toxin gene, csa, which is, to our knowledge, carried by all strains of C. septicum and has been shown to be essential for virulence. Genomic DNAs from a diverse collection of bacterial species, including closely related Clostridium chauvoei, Clostridium carnis, Clostridium tertium as well as several strains of Clostridium perfringens, all failed to produce a positive reaction. An approximate reproducible limit of detection in spiked extracts of at least 10(3) cfu/g of C. septicum was observed for a variety of different sample types. C. septicum levels in broiler chicken field samples estimated from the results of qPCR were statistically correlated to culture based enumerations obtained from those same tissues.
Insights
A new quantitative real-time PCR assay accurately measures Clostridium septicum levels in poultry. This method targets the alpha toxin gene (csa), improving detection for gangrenous dermatitis (GD) research.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Poultry Pathology
Background:
- Clostridium septicum is a significant cause of gangrenous dermatitis (GD) and myonecrotic infections in poultry.
- Accurate enumeration of C. septicum in poultry tissues is challenging due to its swarming growth on agar plates.
- Understanding C. septicum load is crucial for diagnosing and managing GD in poultry flocks.
Purpose of the Study:
- To develop and validate a quantitative real-time PCR (qPCR) assay for precise C. septicum quantification in poultry.
- To target the highly conserved and virulence-associated C. septicum alpha toxin gene (csa) for specific detection.
- To compare qPCR results with traditional culture-based methods for C. septicum enumeration.
Main Methods:
- Development of a species-specific qPCR assay targeting the C. septicum csa gene.
- Testing the assay's specificity against related Clostridium species (e.g., C. chauvoei, C. perfringens).
- Validation of the assay's limit of detection and reproducibility using spiked poultry tissue samples.
Main Results:
- The developed qPCR assay demonstrated high specificity, with no cross-reactivity to closely related bacterial species.
- A reproducible limit of detection of at least 10(3) CFU/g of C. septicum was achieved in various sample types.
- qPCR-based C. septicum quantification in field samples showed a statistically significant correlation with culture-based enumerations.
Conclusions:
- The novel qPCR assay provides a reliable and accurate method for quantifying C. septicum in poultry.
- This molecular tool enhances the ability to study C. septicum pathogenesis and epidemiology in poultry diseases like GD.
- The assay offers a valuable alternative to traditional culture methods, particularly for swarming bacteria.
