The aim of this study was to provide data on the prevalence of serovars of 48 strains of Actinobacillus pleuropneumoniae isolated in Japan from 2006 to 2011, and their antimicrobial resistance profiles (ARPs) by using the disc diffusion method. Of the 48 strains, 10.4% were serovar 1, 60.4% were serovar 2, 14.6% were serovar 5, and 2.1%, 2.1%, 2.1%, and 8.3% were serovars 6, 7, 12, and 15, respectively. We found that 25.0%, 12.5%, 39.6%, 37.5%, 18.8%, and 0% of the strains were resistant to penicillin G, ampicillin, oxytetracycline (OTC), sulfisoxazole, chloramphenicol, and norfloxacin, respectively. The ARPs of each serovar differed, with serovar 1 strains exhibiting multi-antimicrobial resistance to more than three antimicrobials. In contrast, 44.8% of the serovar 2 strains exhibited no resistance, and 34.5% were resistant to only one antimicrobial. All serovar 1, 5, and 15 strains were resistant to OTC, whereas serovar 2 exhibited lower resistance to OTC (6.9%). No significant increase in the prevalence of resistance to these antimicrobials was observed during the last half decade in Japan when compared with published data on the prevalence of resistant strains of A. pleuropneumoniae collected from 1989 to 2005. Regarding the prevalence of serovars, there has been a recent increase in the isolated cases of serovar 15. Discipline: Animal health Additional key words: swine, pleuropneumonia Present address: 2 Kagoshima Chuo Livestock Hygiene Service Center (Higashi-Ichiki, Kagoshima 899-2201, Japan) 3 Kyushu Research Station, National Institute of Animal Health, National Agriculture and Food Research Organization (Kagoshima, Kagoshima 891-0105, Japan) *Corresponding author: e-mail itohiroy@affrc.go.jp Recieved 27 November 2014; accepted 7 May 2015. 11_Kamimura_p073-077.indd 73 2015/11/26 21:15:56 74 JARQ 50 (1) 2016 S. Kamimura et al. Materials and methods Forty-eight strains of A. pleuropneumoniae (isolated from 2006 to 2011 from 47 farms in 11 prefectures in Japan) were sent to our diagnostic laboratory at the National Institute of Animal Health for serotyping. Of the 48 strains, 46 were isolated from different farms. The other two strains of serovar 1 (isolated in 2006 and 2008 at farm A) were subjected to antimicrobial resistance tests to examine ARP changes. A. pleuropneumoniae strain S1421 (= strain ATCC27090) was used as a quality control in each test. A. pleuropneumoniae was grown on tryptic soy agar (Difco, Sparks, MD) supplemented with 5% horse blood and 100 μg/mL β-nicotinamide dinucleotide at 37°C with 5% CO2, and after being suspended in phosphatebuffered saline containing 20% glycerol, it was stored at -80°C until use. Serotyping was carried out with rabbit antisera prepared against serovar reference strains by conducting slide agglutination tests, and when necessary, by immunodiffusion tests (Mittal et al. 1992), along with polymerase chain reaction for genes involved in capsular polysaccharide biosynthesis (Angen et al. 2008, Ito 2010, Zhou et al. 2008). For the antimicrobial resistance tests, we cultured bacterial strains on chocolate agar at 37°C with 5% CO2 as per the Clinical and Laboratory Standards Institute (CLSI) standards and recommendations (CLSI 2013). The resistance of these strains to the following six antimicrobial agents was tested by using the disc diffusion method (Sensi-Disc, Japan Becton Dickinson, Tokyo), as per the CLSI (2013) standards and recommendations: penicillin G (PCG) 10IU, ampicillin (ABPC) 10 μg, oxytetracycline (OTC) 30IU, sulfonamide (sulfisoxazole (SIX)) 250 μg, chloramphenicol (CP) 30 μg, and norfloxacin (NFLX) 10 μg. Each strain was classified as being resistant, susceptible, or intermediate to the antimicrobial agents tested, in accordance with breakpoints proposed by the Comite de l’Antibiogramme de la Societe Francaise Microbiologie (CASFM 2010) as previously described (Vanni et al. 2012), except for SIX and NFLX, for which the criteria of the National Committee for Clinical Laboratory Standards (NCCLS; later renamed CLSI) were applied (NCCLS 2002). Results and discussion Of the 48 strains, 5 (10.4%) were serovar 1, 29 (60.4%) were serovar 2, 7 (14.6%) were serovar 5, 1 (2.1%) was serovar 6, 1 (2.1%) was serovar 7, 1 (2.1%) was serovar 12, and 4 (8.3%) were serovar 15, showing that the most prevalent serovars were 2, 1 and 5, as found in several previous reports (Asawa et al. 1995, Fukuyasu et al. 1996, Morioka et al. 2006, 2008, Yoshimura et al. 2002). Despite the low number of serovar 15 strains isolated in the present study (n=4), serovar 15 has been the fourth most prevalent serovar of strains sent to our diagnostic laboratory for serotyping (Table 1). This prevalence is particularly noteworthy because serovar 15 was not isolated prior to 2008. The rapid rise in the reported cases of serovar 15 strains suggests that this serovar may Table 1. Distribution of antimicrobial resistant strains of Actinobacillus pleuropneumoniae isolated in Japan from 2006 to 2011 Serovar No. of strains (%) PCG ABPC OTC SIX CP NFLX 1 5 (10.4) 3 (60) 3 (60) 5 (100) 4 (80) 4 (80) 0 (0) 2 29 (60.4) 4 (13.8) 1 (3.4) 2 (6.9) 11 (37.9) 5 (17.2) 0 (0) 5 7 (14.6) 3 (42.9) 0 (0) 7 (100) 1 (14.3) 0 (0) 0 (0) 6 1 (2.1) 0 (0) 0 (0) 1 (100) 0 (0) 0 (0) 0 (0) 7 1 (2.1) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 12 1 (2.1) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 15 4 (8.3) 2 (50) 2 (50) 4 (100) 2 (50) 0 (0) 0 (0) Total 48 (100) 12 (25) 6 (12.5) 19 (39.6) 18 (37.5) 9 (18.8) 0 (0) 11_Kamimura_p073-077.indd 74 2015/11/26 21:15:56 75 Serovar and Antimicrobial Resistance of A. pleuropneumoniae have become important relatively recently in Japan, as it has in Australia (Blackall et al. 2002). Table 1 lists the prevalence of resistant strains of the 48 A. pleuropneumoniae strains tested; Table 2 lists the ARPs of the strains where 31.3% (n=15) showed no resistance to any of the six antimicrobials used, and 33.3% (n=16) were resistant to only one antimicrobial, indicating that almost all of the antimicrobials used are effective for swine pleuropneumonia treatment. All of the tested strains were susceptible to NFLX, one of the fluoroquinolones recommended for use as a last-resort antimicrobial (Burch 2008). The strains tested in the present study (Table 1) showed a low prevalence of strains resistant to ABPC (12.5%), which is recommended as the first-choice antimicrobial for swine pleuropneumonia treatment (Burch 2008). A similar low prevalence of ABPC-resistant strains (2.0%-12.4%) in A. pleuropneumoniae was also observed from 1989 to 2005 (Asawa et al. 1995, Fukuyasu et al. 1996, Morioka et al. 2008). Of the 48 strains tested, 12 PCG-resistant strains were identified, half of which were ABPC-resistant and the remaining six were ABPC-susceptible. The underlying reason for the difference in resistance to the two β-lactams (PCG and ABPC) remains to be elucidated. A low prevalence of PCG-resistant strains (4.4%-13.6%) in A. pleuropneumoniae was observed from 1989 to 2000 (Asawa et al. 1995, Fukuyasu et al. 1996, Morioka et al. 2006, Yoshimura et al. 2002). However, the present study showed a slightly increased prevalence of PCG resistance (25.0%) in strains isolated from 2006 to 2011. The two A. pleuropneumoniae serovar 1 strains isolated in 2006 and 2008 at farm A exhibited different ARPs. Specifically, the strain isolated in 2006 was resistant to OTC-SIX-CP, but the 2008 strain was resistant to PCG-ABPC, as well as to OTC-SIX-CP. The ARPs of three other strains isolated from different pigs at farm A in 2006 were further examined in addition to the 48 strains listed in Tables 1 and 2, in order to rule out the possibility that PCG-ABPC-resistant strains may have been already present at farm A in 2006. Consequently, all three strains were found to be resistant to OTC-SIX-CP, but sensitive to PCG-ABPC (data not included in Tables 1 and 2). These results indicate that PCG-ABPC-susceptible clones may have acquired PCG-ABPC resistance or may have been replaced by PCG-ABPC-resistant clones at farm A, resulting in the ineffectiveness of PCG-ABPC for treatment. This observation demonstrates the chronological Table 2. Antimicrobial resistance profiles (ARPs) of 48 Actinobacillus pleuropneumoniae strains tested No. of antimicrobials to which the strains showed resistance ARPs No. of strains (%) Serovar Year isolated 2006 2007 2008 2009 201