Development of a Multiplex Polymerase Chain Reaction Method for <i>cps</i> Typing of <i>Actinobacillus pleuropneumoniae </i>Serovars 1, 2, 5, 7, and 15

Abstract

We developed a multiplex polymerase chain reaction (PCR) method using primers specific to the capsular polysaccharide biosynthesis genes (cps) to type Actinobacillus pleuropneumoniae serovars 1, 2, 5, 7, and 15. This multiplex PCR method may be useful for the typing of serovar 15, which is recently becoming more prevalent, as well as the most prevalent serovars 1, 2, 5, and 7 in Japan. Discipline: Animal health Additional key words: pig, pleuropneumonia, serotyping * Corresponding author: e-mail itohiroy@affrc.go.jp Received 7 October 2014; accepted 24 December 2014. Introduction Actinobacillus pleuropneumoniae is an etiologic agent of porcine pleuropneumonia that causes serious economic losses in the pig-rearing industry (Gottschalk 2012). Fifteen serovars of A. pleuropneumoniae have been identified to date, mainly based on the antigenic diversity of their capsular polysaccharides (Blackall et al. 2002, Perry et al. 1990). The prevalent local serovars vary among countries (Gottschalk 2012); in Japan, serovars 2 (59%), 1 (28%), 5 (8%), and 7 (3%) account for approximately 98% of the strains isolated from pigs (Asawa et al. 1995, Fukuyasu et al. 1991, 1996, Kume & Nakai 1988, Morioka et al. 2006, 2008, Suzuki et al. 1989, Yoshimura et al. 2002). However, the incidence of isolated cases of serovar 15, which is the most recently identified serovar (Blackall et al. 2002), has been recently increasing in Japan (Ito 2013, Morioka et al. 2008). Because the virulence of strains differs depending on their serovars and differences in serovars affect the effectiveness of vaccines (Gottschalk 2012), serotyping is widely performed in veterinary diagnostic laboratories. However, cross-reactions are often observed among different serovars, for example, among serovars 1, 9, and 11 (Gottschalk 2012), serovars 3, 6, 8, and 15 (Gottschalk 2012), serovars 4 and 7 (Gottschalk 2012), and serovars 7 and 15 (Blackall et al. 2002, Koyama et al. 2007), preventing the accurate and rapid typing of field strains. In the last decade, serovar-specific polymerase chain reaction (PCR) typing methods based on the sequence variation in genes involved in capsular polysaccharide biosynthesis (cps) have been developed for serovars 1-3, 5-8, 10, 12, and 15 (Angen et al. 2008, Bosse et al. 2014, Ito 2010, Jessing et al. 2003, Lo et al. 1998, Schuchert et al. 2004, Turni et al. 2014, Zhou et al. 2008). These PCR typing methods facilitate the reliable typing of A. pleuropneumoniae serovars without cross-reactions and mistyping. In the present study, we developed a multiplex PCR method for cps typing of A. pleuropneumoniae serovars 1, 2, 5, and 7, which are the most prevalent in Japan (Asawa et al. 1995, Fukuyasu et al. 1991, 1996, Kume & Nakai 1988, Morioka et al. 2006, 2008, Suzuki et al. 1989, Yoshimura et al. 2002), as well as serovar 15, isolated cases of which have been increasing (Ito 2013, Morioka et al. 2008). Materials and methods We used the A. pleuropneumoniae serovar reference strains serovar 1, 4074; serovar 2, S1536; serovar 3, S1421; serovar 4, M62; serovar 5a, K17; serovar 6, Femo, serovar 7, WF83, serovar 8, 405; serovar 9; CVJ13261; serovar 10, D13039; serovar 11; 56153; serovar 12, 8329; serovar 13, N273; serovar 14, 4906; and serovar 15, HS143, to determine suitable conditions for multiplex PCR. Seventy-