p.m. Actinobacillus pleuropneumoniae isolates were either obtained from existing collections maintained in our

University, or kindly provided by Dr Huanchen Chen (Huazhong Agricultural University, Wuhan, China) and Dr Youxiang Diao (Shandong Agricultural University, Tai’an, China). The chromosomal DNA from A. pleuropneumoniae was extracted using the AxyPrep Bacterial Genomic DNA Miniprep kit (Axygen) according to the manufacturer’s instructions. RDA was performed using a previously check details described method (Lisitsyn & Wigler, 1993). The following adapters and primers were used for RDA (listed in Table 2): R-Bgl 12/R-Bgl 24, J-Bgl 12/J-Bgl 24, and N-Bgl 12/N-Bgl 24. Briefly, the DNA fragments were digested with Sau3AI (TaKaRa), the R-Bgl 12/R-Bgl 24 adapters were ligated to the digested DNA to be used as the tester. The first differential product (DP1) was obtained by performing hybridization (20 h at 67 °C) with a driver : tester ratio

of 100 : 1, and this product was amplified by PCR with an R-Bgl 24 primer. The second (DP2) and third (DP3) differential products were generated by ligating the N-Bgl and J-Bgl adapters to the tester in the second and third rounds of subtractive hybridization, with driver : tester ratios of 400 : 1 and 8000 : 1, respectively. The differential DNA fragments for CVCC259 were obtained using CVCC259 as the tester and CVCC261 as the driver; this combination was designated as ‘a.’ Similarly, the differential DNA fragments for CVCC261 were obtained using CVCC261 as the tester and CVCC259 as the driver; this combination was designated as ‘b. The DP3 differential products were 5-FU manufacturer purified using the

Qiaquick PCR purification kit (Qiagen) and ligated into the pGEM-T vector (Promega). The RDA library was constructed by transforming the ligation mixture into competent Escherichia coli DH5α cells (TaKaRa). The inserts were sequenced by the BGI-GBI Biotech Company (Beijing, China). The blastn program was used to locate the sequence similarity nearly in the GenBank database. The differential nature of the DNA sequences was confirmed using a novel application of the reverse Southern hybridization procedure (Lancashire et al., 2007). The differential DNA fragments were successively spotted onto a nylon membrane. The membrane was baked at 120 °C for 30 min. The probes were prepared using 6 μg of the Sau3AI-digested genomic DNA obtained from the CVCC259 and CVCC261 strains and separately labeled using digoxigenin (DIG)-High Prime (Roche). Nonradioactive labeling, hybridization, and detection were performed using the DIG-High Prime DNA Labeling and Detection Starter Kit (Roche) according to the manufacturer’s instructions. Because all the amplified differential sequences contained the J-Bgl 24 primer, the J-Bgl 24 primer was considered as the negative control. To further characterize the differential DNA sequences, we designed specific primers using the primer 5.0 software (listed in Table 2).