pcr specific for Actinobacillus pleuropneumoniae serotype 3

Abstract

Serotypes 3 and 8 of Actinobacillus pleuropneumoniae, the aetiological agent of porcine pleuropneumonia, have been reported to predominate in the UK. Direct serotyping of isolates of the organism is typically determined by the immunological reactivity of rabbit serum to its surface polysaccharides, but the method has limitations, for example, cross-reactions between serotypes 3, 6 and 8. This study describes the development of a serotype 3-specific pcr, based on the capsule locus, which can be used in a multiplex format with the organism's specific gene apxIV. The pcr test was evaluated on 266 strains of A pleuropneumoniae and 121 strains of other organisms, including all the major respiratory bacterial pathogens of pigs. The test was highly specific and sensitive and should be useful for differentiating strains of serotypes 3, 6 and 8, and in seroprevalence and epidemiological surveys in regions where serotype 3 is prevalent, such as the UK.

Multiplex PCR That Can Distinguish between Immunologically Cross- Reactive Serovar 3, 6, and 8 Actinobacillus pleuropneumoniae Strains

“…), we were unable to combine our serovar 3-and apxIV-specific primers with those previously described for serovars 6 and 8 (17,45). Here we describe a new set of serovar 3-and serovar 8-specific primers that can be multiplexed with those described for serovar 6 (17) and apxIV (53) to enable a highly sensitive and specific test that can discriminate between the problematic cross-reactive serovar 3, 6, and 8 strains in a single PCR.…”

“…The apxIV gene is specific to A. pleuropneumoniae and can be used to verify test strains to the species level (43). In the context of a multiplex PCR, the use of apxIV also allows evaluation of the integrity of genomic DNA (53). Unfortunately, despite exhaustive variations in experimental PCR parameters (buffer conditions, magnesium salt concentrations, annealing temperatures, primer concentrations, etc.…”

“…Although serovar 6-and serovar 8-specific primers have been described as part of other multiplex PCRs (17,45), to date, there has not been a description of a multiplex PCR that can discriminate between the cross-reactive serovar 3, 6, and 8 strains. In our previous study we described a serovar 3-specific apxIV multiplex PCR (53). The apxIV gene is specific to A. pleuropneumoniae and can be used to verify test strains to the species level (43).…”

“…In our previous study (53), to obtain the serovar 3-specific sequence, we exploited the conserved nature of gene loci involved in capsule expression that occurs in many gram-negative bacteria including A. pleuropneumoniae (51,52). An oligonucleotide, AP5C, that annealed to a conserved consensus sequence in the capsular export region of A. pleuropneumoniae (45), combined with the arbitrary primer ARB6, resulted in a 4-kb PCR amplicon that, after DNA sequencing of 2,732 bp, was used to identify two serovar 3-specific primers.…”

“…In the present study, the 2,732 bp of DNA sequence available (GenBank accession number EU090171) was reanalyzed, and two further potential serovar 3-specific primers, AP3NR (5Ј-AAC AAA TAA AGT TGC TCG AAA GTA-3Ј) and AP3NF (5Ј-TTT GCG CTG TAG TGC TCC AAT-3Ј), were identified. In initial experiments, AP3NR and AP3NF were multiplexed with the primers APXIVA1 (5Ј-TTA TCC GAA CTT TGG TTT AGC C-3Ј) and APXIVA3 (5Ј-CAT ATT TGA TAA AAC CAT CCG TC-3Ј) specific for apxIV (53), the primers AP6F (5Ј-AAC CAC TCA CTT TCC ACA TTA G-3Ј) and AP6R (5Ј-AAT CGG AAG GTT TTG GTC TCG TG-3Ј) specific for serovar 6 used by Jessing et al (17), and the primers AP8NR (5Ј-GAT TAA ACT GGT CCG TCG AAA TG-3Ј) and AP8NF (5Ј-TTA GTT GCG CAA ACG GCT TTT GAA-3Ј) specific for serovar 8, which were designed from the available sequence of the cps8ABC genes (accession number AY356527). The serovar 8 primers described by Shuchert et al (45) were not used since these primers result in a PCR band of 970 bp, which is similar in size to that for serovar 3 (918 bp).…”