Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Evaluation of a multiplex PCR to identify and serotype Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15 C. Turni1, R. Singh1, M.A. Schembri2 and P.J. Blackall1 1 Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Dutton Park, Qld, Australia 2 School of Chemistry and Molecular Biosciences, and Australian Infectious Diseases Research Centre, The University of Queensland, St Lucia, Qld, Australia

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.

Keywords Actinobacillus pleuropneumoniae, identification, multiplex PCR, serotyping, validation. Correspondence Conny Turni, Queensland Alliance for Agriculture and Food Innovation, Ecosciences Precinct, GPO Box 46, Brisbane, Qld 4101, Australia. E-mail: [email protected] 2014/0492: received 11 March 2014, revised 20 May 2014 and accepted 20 May 2014 doi:10.1111/lam.12287

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.

Introduction Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia (Taylor 1999). This disease contributes to substantial economic losses in the swine industry worldwide, with 30–60% of pigs in conventional herds carrying A. pleuropneumoniae in the nasal cavity or in the tonsils (Taylor 1999). There are a total of 15 serovars of A. pleuropneumoniae recognized, with the current structure being an amalgamated scheme that includes both nicotinamide (NAD)-dependent and NAD-independent biovars of A. pleuropneumoniae (Blackall et al. 2002). There is considerable value in the capacity to serotype A. pleuropneumoniae since serovars are known to show considerable difference in virulence (Rosendal et al. 1985; Rogers et al. 362

1990). Further, killed whole-cell pleuropneumonia vaccines provide protection only against those serovars present in the vaccine (Nielsen 1976, 1984). A number of serological assays have been developed for serotyping A. pleuropneumoniae. While there are rapid methods such as slide agglutination, in some cases, specificity is compromised due to cross-reactivity (Mittal 1990). Other more time-consuming methods such as immunodiffusion and indirect haemagglutination can be used for definitive serotyping (Nielsen and O’Connor 1984). A key issue with all serological tests is the need for high titre and highly specific anti-sera—a requirement that often limits serotyping to central reference laboratories. Alternatives to the conventional serotyping have been published (Gram et al. 2000; Sthitmatee et al. 2003). In recent years, PCR assays that detect the genes associated

Letters in Applied Microbiology 59, 362--369 © 2014 The Society for Applied Microbiology

PCR serotyping of A. pleuropneumoniae

C. Turni et al.

with capsular lipopolysaccharides have been developed to identify some serovars of A. pleuropneumoniae, as these polysaccharides are known to be the primary serovar-specific antigen (Inzana and Mathison 1987). Several multiplex PCRs have been published that can identify serovars 2, 5 and 6 (Jessing et al. 2003) or 1, 2 and 8 (Schuchert et al. 2004) or 1, 7 and 12 (Angen et al. 2008) or 3, 6 and 8 (Zhou et al. 2008). In Australia, the most prevalent serovars are serovars 1, 5, 7 and 15 with an increase of serovar 12 in recent years (Turni et al. 2013). To enable molecular identification of the most prevalent serovars in one assay, the current work reports on the validation of a multiplex PCR that combines existing assays for species specificity to include serovars 1, 5, 7 and 12 as well as a novel assay specific for serovar 15. Results and discussion Five of the six sets of primers used in the multiplex PCR developed in this study have a well established capacity for specificity for either the species (Gram and Ahrens 1998) or for serovars 1, 7 and 12 (Angen et al. 2008) and serovar 5 (Lo et al. 1998). However, in the multiplex PCR developed in this work, minor changes were introduced in the PCR conditions and the sequences of the serovar 12 primers were modified. The changes in PCR conditions involved alteration of the annealing temperature (increase in temperature from 63 to 66°C) and the duration of each of the stages of the PCR cycle. Hence, a validation of the multiplex PCR was necessary. A key innovation of this multiplex PCR is the capacity to recognize serovar 15. The main serovars of Australian A. pleuropneumoniae isolates identified over an 11 years period (2002–2013) were serovars 15 (33%), 7 (28%), 5 (18%), serovar 1 (9%) and serovar 12 (3%), with 4% of the isolates unable to be assigned to a serovar (Turni et al. 2013). However, the sequence of the serovar-specific DNA region involved in biosynthesis of the capsular polysaccharides (cps genes), which are the targets for the published serotyping PCRs (Lo et al. 1998; Jessing et al. 2003; Angen et al. 2008; Zhou et al. 2008), was not known for serovar 15. Schuchert et al. (2004) described a conserved DNA region in the capsular polysaccharide export regions (cpxDCBA), which is adjacent to the capsular polysaccharide biosynthesis region (cpsABCD). With cpxAF and cpxAR primers designed for the conserved region of serovar 5, they amplified isolates of serovar 1 to serovar 12 (Schuchert et al. 2004). This region was also conserved in serovar 15 and was the starting point for the subsequent chromosomal walking to identify primer sequences specific for serovar 15. All 15 reference strains and all 411 field isolates of A. pleuropneumoniae yielded a 951 bp band specific for

A. pleuropneumoniae, while none of the 26 nontarget taxa yielded this species-specific band in the multiplex PCR. Importantly, the 16S rRNA PCR revealed that all the DNA from all of the nontarget taxa was suitable for PCR amplification. The species specificity of the Gram and Ahrens (1998) PCR, which targets the omlA gene, has now been confirmed in a number of subsequent studies, for example Angen et al. (2008). The only other organism known to give the same size amplicon in the omlA PCR is Actinobacillus genomospecies 1 (Angen et al. 2008). As Actinobacillus genomospecies 1 is associated with horses (Christensen et al. 2002), the false-positive reaction of this taxon is not a diagnostic problem. Three of the nontarget taxa yielded a product similar to serovar 12 (557 bp) and serovar 15 (269 bp) serovarspecific bands in the multiplex PCR—A. capsulatus (an c. 270 bp band and a very faint c. 580 bp band), A. indolicus (type strain—an c. 560 bp band) and A. suis (an c. 270 bp band and a very faint c. 690 bp band). None of these products were close to the size of the species-specific product expected in the PCR (951 bp) and therefore did not present a problem in terms of interpretation (Table 2, Fig. 1). The original multiplex PCR for serovars 1, 7 and 12 as developed by Angen et al. (2008) included four of the six primer sets used in the current study. Angen et al. (2008) performed a similar specificity examination using many of the same nontarget reference strains as in this study. In contrast to the current findings of three taxa yielding nonspecific bands, Angen et al. (2008) reported only that four A. lignieresii strains yielded an amplicon identical to that of serovar 7, while another isolate produced an amplicon identical to that of serovar 1. The fact that Angen et al. (2008) examined 45 A. lignieresii field isolates and the reference strain, while the current study only examined the reference strain may explain the difference in the two studies in terms of the results with A. lignieresii. It is more difficult to explain why nonspecific bands in the reference strains for A. capsulatus, A. indolicus and A. suis were detected in the current study that were not reported by Angen et al. (2008) and Lo et al. (1998), except that the reverse primer for serovar 15 was similar to the sequence of A. suis. The changed PCR conditions and the addition of the serovar 15 primers to the reaction may account for these observations. While the multiplex PCR did yield minor PCR products with some of the nontarget species, this did not affect identification at the species level. All of the nonspecific bands were

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

The aim of this study was to validate a multiplex PCR for the species identification and serotyping of Actinobacillus pleuropneumoniae serovars 1, 5, ...
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