Use of a nested PCR-enzyme immunoassay with an internal control to detect <it>Chlamydophila psittaci </it>in turkeys

• Main Authors: Goddeeris Bruno M, Volckaert Guido, Messmer Trudy O, Verminnen Kristel, Van Loock Marnix, Vanrompay Daisy
• Format: Article
• Language: English
• Published: BMC 2005-09-01
• Series: BMC Infectious Diseases
• Online Access: http://www.biomedcentral.com/1471-2334/5/76

<p>Abstract</p> <p>Background</p> <p>Laboratory diagnosis of <it>Chlamydophila psittaci</it>, an important turkey respiratory pathogen, is difficult. To facilitate the diagnosis, a nested PCR-enzyme immunoassay (PCR-EIA) was developed to detect the <it>Cp. psittaci </it>outer membrane protein A (<it>ompA</it>) gene in pharyngeal swabs.</p> <p>Methods</p> <p>The fluorescein-biotin labelled PCR products were immobilized on streptavidin-coated microtiter plates and detected with anti-fluorescein peroxidase conjugate and a colorimetric substrate. An internal inhibition control was included to rule out the presence of inhibitors of DNA amplification. The diagnostic value of the <it>ompA </it>nested PCR-EIA in comparison to cell culture and a 16S-rRNA based nested PCR was assessed in pharyngeal turkey swabs from 10 different farms experiencing respiratory disease.</p> <p>Results</p> <p>The sensitivity of the nested PCR-EIA was established at 0.1 infection forming units (IFU). Specificity was 100%. The <it>ompA </it>nested PCR-EIA was more sensitive than the 16S-rRNA based nested PCR and isolation, revealing 105 out of 200 (52.5%) positives against 13 and 74 for the latter two tests, respectively. Twenty-nine (23.8%) out of 122 <it>ompA </it>PCR-EIA negatives showed the presence of inhibitors of DNA amplification, although 27 of them became positive after diluting (1/10) the specimens in PCR buffer or after phenol-chloroform extraction and subsequent ethanol precipitation.</p> <p>Conclusion</p> <p>The present study stresses the need for an internal control to confirm PCR true-negatives and demonstrates the high prevalence of chlamydiosis in Belgian turkeys and its potential zoonotic risk. The <it>ompA </it>nested PCR-EIA described here is a rapid, highly sensitive and specific diagnostic assay and will help to facilitate the diagnosis of <it>Cp. psittaci </it>infections in both poultry and man.</p>