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Journal of Clinical Microbiology, November 2003, p. 5085-5093, Vol. 41, No. 11
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.11.5085-5093.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Evaluation and Field Validation of PCR Tests for Detection of Actinobacillus pleuropneumoniae in Subclinically Infected Pigs

Nahuel Fittipaldi,1,2 André Broes,2,3 Josée Harel,1,2 Marylène Kobisch,4 and Marcelo Gottschalk1,2*

Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada J2S 7C6,1 Canadian Research Network on Bacterial Pathogens of Swine,2 Centre de Développement du Porc du Québec Inc., Sainte-Foy, Québec, Canada G1V 4M7,3 Agence Française de Sécurité Sanitaire des Aliments, Laboratoire d'Études et de Recherches Avicoles et Porcines, Unité de Mycoplasmologie-Bactériologie, 22440 Ploufragan, France4

Received 15 April 2003/ Returned for modification 16 May 2003/ Accepted 18 August 2003

Eight PCR tests were evaluated for their abilities to detect Actinobacillus pleuropneumoniae in swine tonsils. At first they were compared regarding their specificities by using A. pleuropneumoniae and related bacterial species and their analytical sensitivities by using tonsils experimentally infected in vitro. PCRs were carried out both directly with tonsil homogenates (direct PCR) and after culture of the sample (after-culture PCR). Most tests demonstrated good specificities; however, some tests gave false-positive results with some non-A. pleuropneumoniae species. High degrees of variation in the analytical sensitivities among the tests were observed for the direct PCRs (109 to 102 CFU/g of tonsil), whereas those of most of the after-culture PCRs were similar (102 CFU/g of tonsil). In a second phase, the effects of sample storage time and storage conditions were evaluated by using tonsils from experimentally infected animals. Storage at -20°C allowed the detection of the organism for at least 4 months. Finally, the omlA PCR test described by Savoye et al. (C. Savoye et al., Vet. Microbiol. 73:337-347, 2000) and the commercially available Adiavet App PCR test were further validated with field samples. Their effectiveness was compared to those of standard and immunomagnetic separation-based methods of bacterial isolation. In addition, a comparison of tonsil biopsy specimens (from living animals) and whole tonsils (collected at the slaughterhouse) was also conducted. A. pleuropneumoniae was neither isolated nor detected by PCR from a herd serologically negative for A. pleuropneumoniae. PCR was more sensitive than the standard isolation method with whole tonsils from three infected herds. After-culture PCR offered the highest degree of sensitivity (93 and 83% for the omlA and Adiavet App PCRs, respectively). The PCR detection rate was higher with whole tonsils than with tonsil biopsy specimens. Good agreement ({kappa} = 0.65) was found between the presence of A. pleuropneumoniae in tonsils and the individual serological status of the animals.

* Corresponding author. Mailing address: GREMIP, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 rue Sicotte, CP 5000, St-Hyacinthe, Québec, Canada J2S 7C6. Phone: (450) 773-8521, ext. 8374. Fax: (450) 778-8108. E-mail: marcelo.gottschalk{at}umontreal.ca.

Journal of Clinical Microbiology, November 2003, p. 5085-5093, Vol. 41, No. 11
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.11.5085-5093.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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