Proposal of Actinobacillus pleuropneumoniae serovar 19, and reformulation of previous multiplex PCRs for capsule-specific typing of all known serovars

Rapid Detection and Typing of Actinobacillus pleuropneumoniae Serovars Directly From Clinical Samples: Combining FTA® Card Technology With Multiplex PCR

“…For optimization of FTA ® card processing conditions, single-plex PCR reactions were prepared with either a PCR kit containing HotStarTaq DNA Polymerase (Qiagen Ltd., Cat #206152) or DreamTaq HotStart DNA Polymerase (Thermo Fisher UK, Loughborough, UK, Cat #K9011) in 50 μl total reaction volumes (prepared according to manufacturers' protocols), with serovar 19-specific primers ( 12 ) at a final concentration of 0.2 μM of each, and one prepared 3-mm sample disc per tube. Multiplex PCRs for A. pleuropneumoniae identification and typing were performed with the previously published APP-mPCR1 and APP-mPCR2 primer sets ( 12 ), using the Qiagen Multiplex Plus PCR Kit (Qiagen Ltd., Cat #206152), in 50 μl total reaction volumes consisting of 25 μl 2X Master Mix (containing HotStarTaq DNA Polymerase), 0.2 μM of each primer, 5 μl 10x CoralLoad gel-tracking dye (Qiagen Ltd.), and either one prepared sample disc or 4 μl gDNA. Reactions were run on a T100 Thermal Cycler (Bio-Rad, Cressier, Switzerland, Cat #1861096) for 10 min at 95°C, followed by 30 cycles of 95°C for 15 s, 60°C for 90 s, and 72°C for 150 s, with a final extension at 68°C for 10 min.…”

“…Although all are capable of causing pleuropneumonia, some serovars are considered more pathogenic than others (8). Furthermore, regional differences in the distribution and prevalence of specific serovars can change over time, and since 2015, the number of known serovars of A. pleuropneumoniae has increased from 16 to 19, with the identification of novel cps loci in previously non-typable isolates (9)(10)(11)(12). In the UK, serovar 8 has accounted for >70% of clinical isolates for more than a decade (13,14), with serovars 2, 6, 7, and 12 also occasionally isolated.…”

“…Additionally, FTA R cards have been implemented in the diagnosis of numerous veterinary bacterial pathogens (21)(22)(23)(24)(25), including members of the Pasteurellaceae (16,26), but not (to our knowledge) A. pleuropneumoniae. Therefore, in order to reduce biosafety concerns and enable rapid and affordable diagnostics of this important swine pathogen, we have developed and validated an FTA R card protocol for processing clinical samples or bacterial cultures, which can be coupled to our previously described APP-mPCRs (10,12) for molecular detection and typing of A. pleuropneumoniae.…”

“…A. pleuropneumoniae serotyping is important for implementation and monitoring of vaccination programs, as the current vaccines (bacterins) provides little heterologous protection and the efficacy depends on correct coverage of serovar(s) circulating in the region at a given time. Recently, we validated two multiplex PCRs (APP-mPCR1 and APP-mPCR2) for detection and molecular typing of all 19 A. pleuropneumoniae serovars using DNA extracted from cultured bacteria ( 7 , 12 ).…”

Efficacy in Reduction of Lung Lesions of a Toxin Expressing Whole-cell Vaccine Against Multiple Serovars of Actinobacillus Pleuropneumoniae in Growing Pigs

“…So far nineteen A. pleuropneumoniae serovars have been classi ed worldwide 41 . However, as the difference between serovar 9 and 11 is only one amino acid in the complete CPS loci and they have identical toxin pro les (ApxI, ApxII), these two serovars can be considered as one: serovar 9/11 42 .…”