Isolation of Actinobacillus pleuropneumoniae serotype 2 by immunomagnetic separation

In Denmark porcine pleuropneumonia is most frequently caused by Actinobacil lus pleuropneumoniae serotype 2 (60%). Isolation of A. pleuropneumoniae fro m nasal cavities or tonsils from carrier animals is complicated due to the mixed bacterial flora present. An immunomagnetic separation technique (IMS) using immunomagnetic beads (Dynabeads(R)) was developed for isolation of A . pleuropneumoniae serotype 2 from pure cultures and from heterogeneous sus pensions. Different coating and washing procedures were evaluated in pure a nd mixed cultures using polyclonal (PAb) and monoclonal antibodies. The hig hest reisolation yield was achieved when the beads were coated with 1.5 mug PAb IgG/10(7) beads. After washing the beads for four times 9-24% of the b acteria could be reisolated depending on the amount of IgG attached to the beads and the number of beads used. The recovery was increased to 19-61% wh en only two washing steps were performed. The IMS was further evaluated usi ng dilutions of A. pleuropneumoniae with added Pasteurella multocida (10(9) CFU/ml). After two washing steps 15% of the A. pleuropneumoniae cells and no P. multocida was reisolated. A detection limit of 10 CFU/ml was found in this heterogeneous suspension. No significant difference was observed when comparing the recovery of A. pleuropneumoniae from pure culture, from mixe d cultures and from artificially inoculated tonsils. From 12 pigs inoculate d with an aerosol of A. pleuropneumoniae serotype 2 the bacterium could not be detected from the nasal cavity or tonsils by cultivation or PCR 6 weeks later. By using IMS A. pleuropneumoniae serotype 2 could be reisolated fro m the tonsils of three pigs. The LMS method represents a valuable tool for isolation of A. pleuropneumoniae from tissue samples. (C) 2001 Elsevier Sci ence B.V. All rights reserved.