Development of Multilocus Sequence Typing (MLST) of Actinobacillus Pleuropneumniae

Abstract

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae (AP) infection leads to considerable financial loss in the swine industry worldwide. Multilocus sequence typing (MLST), a definitive molecular typing technique that is highly reproducible and is capable of comparing data across laboratories, has not been studied on AP. Therefore, the aim of this work is to develop a MLST assay to characterize AP isolates collected from Taiwan. A total of 85 AP isolates collected from pleuropneumonia of diseased pigs with respiratory symptoms and seven reference isolates from other countries were included for comparison. Seven housekeeping genes (recF, gly, rho, tpi, pyk, recN, and rpo) were selected for sequencing. Capsule types that determine serotypes and Apx toxins were analyzed by the polymerase chain reaction (PCR). The sequencing results showed that the 7 housekeeping genes differentiated the 92 isolates into 14 sequence types that belonged to three major clonal complexes and five singletons by eBURST. The 85 isolates assigned to detection of capsule types and Apx toxins showed that serotype 1 with Apx I, II, IV (55.3%) followed by serotype 5 with Apx I, II, IV (29.4%) were the most prevalent in Taiwan. Notably, serotype 15 was identified for the first time. Clonal complexes based on ST profiles from MLST analysis were highly associated with the distribution of capsule types, suggesting that MLST scheme was sufficient to identify DNA samples directly from AP. Therefore, MLST might be a useful tool for identification and further epidemiological assay of AP.