Evaluation of a multiplex PCR to identify and serotype Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15

Abstract

The aim of this study was to validate a multiplex PCR for the species identification and serotyping of Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15. All 15 reference strains and 411 field isolates (394 from Australia, 11 from Indonesia, five from Mexico and one from New Zealand) of A. pleuropneumoniae were tested with the multiplex PCR. The specificity of this multiplex PCR was validated on 26 non-A. pleuropneumoniae species. The multiplex PCR gave the expected results with all 15 serovar reference strains and agreed with conventional serotyping for all field isolates from serovars 1 (n = 46), 5 (n = 81), 7 (n = 80), 12 (n = 16) and serovar 15 (n = 117). In addition, a species-specific product was amplified in the multiplex PCR with all 411 A. pleuropneumoniae field isolates. Of 25 nontypeable field isolates only two did not yield a serovar-specific band in the multiplex PCR. This multiplex PCR for serovars 1, 5, 7, 12 and 15 is species specific and capable of serotyping isolates from diverse locations. Significance and impact of the study: A multiplex PCR that can recognize serovars 1, 5, 7, 12 and 15 of A. pleuropneumoniae was developed and validated. This novel diagnostic tool will enable frontline laboratories to provide key information (the serovar) to guide targeted prevention and control programmes for porcine pleuropneumonia, a serious economic disease of pigs. The previous technology, traditional serotyping, is typically provided by specialized reference laboratories, limiting the capacity to respond to this key disease.

Molecular serotyping and antimicrobial resistance profiles ofActinobacillus pleuropneumoniaeisolated from pigs in South Korea

“…The distribution of A. pleuropneumoniae serotypes varied by region and with time. For example, in Australia, the most prevalent serotypes are 1, 5, 7, and 15 with a recent increase in serotype 12 (Turni et al 2014). Serotypes 2 and 9 are the most prevalent in the Czech Republic (Kucerova et al 2011), and serotypes 2 and 4 are dominant in Spain (Guti errez-Mart ın et al 2006).…”

“…In recent years, PCR (polymerase chain reaction) assays for detecting genes associated with capsular polysaccharides have been developed to identify A. pleuropneumoniae serotypes (Jessing et al 2003;Boss e et al 2014;Turni et al 2014). Sequence differences in the middle region of the A. pleuropneumoniae outer membrane lipoprotein omlA gene divide the A. pleuropneumoniae serotypes into four distinct groups, and this sequence variation has been used for speculating serotypes (Gram et al 2000;Angen et al 2008).…”

“…Other more time-consuming methods such as immunodiffusion and indirect hemagglutination can be used for definitive serotyping (Nielsen & O'Connor 1984). A critical issue with all serological testing is the need for high titers and highly specific anti-sera, which are requirements that often limit the performance of serotyping to central reference laboratories (Turni et al 2014).…”

Real-time quantitative PCR for detection and identification of Actinobacillus pleuropneumoniae serotype 2

“…In recent years, several PCR assays have been described for detection of specific serotypes of A. pleuropneumoniae (1,8,14,15); however, only one quantitative PCR based on SYBR Green was evaluated for detection of serotype 2 (11). The use of TaqMan probes ensures higher reaction specificity and does not require additional analysis of melting curves to identify nonspecific amplification products (e.g.…”

“…In addition, some serotypes cannot be correctly identified due to cross-reactions observed during serotyping with antisera (12). To overcome these problems, several molecular techniques have been developed to identify A. pleuropneumoniae serotypes from pure cultures (1,2,8,9,14,15). PCR tests for individual identification of A. pleuropneumoniae serotype 2 have also been described (8,9,11,14), but no real-time quantitative TaqMan probe-based PCR has been developed.…”