title: Use of a nested PCR-enzyme immunoassay with an internal control to detect Chlamydophila psittaci in turkeys.
abstract: BACKGROUND
  Laboratory diagnosis of Chlamydophila psittaci, an important turkey respiratory pathogen, is difficult. To facilitate the diagnosis, a nested PCR-enzyme immunoassay (PCR-EIA) was developed to detect the Cp. psittaci outer membrane protein A (ompA) gene in pharyngeal swabs.
  
  
  METHODS
  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 ompA 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.
  
  
  RESULTS
  The sensitivity of the nested PCR-EIA was established at 0.1 infection forming units (IFU). Specificity was 100%. The ompA 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 ompA 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.
  
  
  CONCLUSIONS
  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 ompA nested PCR-EIA described here is a rapid, highly sensitive and specific diagnostic assay and will help to facilitate the diagnosis of Cp. psittaci infections in both poultry and man.
authors.affiliation: Department of Biosystems, Catholic University of Leuven, Kasteelpark Arenberg 30, 3001 Heverlee, Belgium. marnix.van.loock@pandora.be
authors.forename: Marnix
authors.initials: M
authors.lastname: Van Loock
authors.forename: Kristel
authors.initials: K
authors.lastname: Verminnen
authors.forename: Trudy O
authors.initials: TO
authors.lastname: Messmer
authors.forename: Guido
authors.initials: G
authors.lastname: Volckaert
authors.forename: Bruno M
authors.initials: BM
authors.lastname: Goddeeris
authors.forename: Daisy
authors.initials: D
authors.lastname: Vanrompay
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology; D002691:Chlamydophila psittaci / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D007124:Immunoenzyme Techniques / Q000379:methods / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D011201:Poultry Diseases / Q000175:diagnosis* / Q000382:microbiology*; D009956:Psittacosis / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D014422:Turkeys / Q000382:microbiology*
chemical_list: D001425:Bacterial Outer Membrane Proteins; C078167:OMPA outer membrane proteins