Author
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Wise, Mark |
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Siragusa, Gregory |
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Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 1/26/2005 Publication Date: 7/1/2005 Citation: Wise, M., Siragusa, G.R. 2005. Semi-Quantitative Detection of Clostridium Perfringens In The Broiler Fowl Gastrointestinal Tract by Real-Time PCR. Applied and Environmental Microbiology. 71:3911-3916. Interpretive Summary: Clostridium perfringens is an important cause of food-borne disease, as well as the causative agent of necrotic enteritis in chickens. As such, it has a dual role as a human pathogen, as well as an agent potentially detrimental to animal health. C. perfringens-associated necrotic enteritis in the boiler chicken is a potentially fatal disorder that is routinely controlled with preventative antibiotics added to the feed or water. However, the use of preventative growth-promoting antibiotics in agriculture may result in strains of bacteria resistant to antibiotics used to treat humans. Since one result of not feeding growth promoting antibiotics is a higher rate of poultry necrotic enteritis, the poultry industry is searching for antibiotic alternatives in order to reduce the amount of growth-promoting antibiotics used to raise broiler chickens. Present methods to count numbers of C. perfringens in the intestinal tract of broiler chickens are laborious and time consuming. Therefore we developed an easy and rapid method to quantify this species in the broiler chicken gastrointestinal tract. The assay, based on a technique known as quantitative real-time PCR, is a DNA-based test that detects only C. perfringen. We have used this test to determine the levels of C. perfringens in broiler intestinal contents from necrotic and healthy chickens reared without growth promoting antibiotics. Comparative results indicated that the new procedure correlated well with the standard method of plate counts, especially from samples taken from the middle region of the gastrointestinal tract. This assay will be useful to study the intestinal microorganisms of poultry in the search for antibiotic alternatives. Technical Abstract: Strains of Clostridium perfringens are a frequent cause of food-borne disease, gas gangrene, and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a semi-quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of Cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA, and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 cfu/g ileal material, but only about 104 cfu/g in cecal samples. The decreased sensitivity of the cecal samples was due to the presence of unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract. |
