A novel quantitative real-time polymerase chain reaction method for detecting toxigenic Pasteurella multocida in nasal swabs from swine
© The Author(s) 2016
Received: 20 April 2016
Accepted: 25 November 2016
Published: 1 December 2016
Progressive atrophic rhinitis (PAR) in pigs is caused by toxigenic Pasteurella multocida. In Switzerland, PAR is monitored by selective culture of nasal swabs and subsequent polymerase chain reaction (PCR) screening of bacterial colonies for the P. multocida toxA gene. A panel of 203 nasal swabs from a recent PAR outbreak were used to evaluate a novel quantitative real-time PCR for toxigenic P. multocida in porcine nasal swabs.
In comparison to the conventional PCR with a limit of detection of 100 genome equivalents per PCR reaction, the real-time PCR had a limit of detection of 10 genome equivalents. The real-time PCR detected toxA-positive P. multocida in 101 samples (49.8%), whereas the conventional PCR was less sensitive with 90 toxA-positive samples (44.3%). In comparison to the real-time PCR, 5.4% of the toxA-positive samples revealed unevaluable results by conventional PCR.
The approach of culture-coupled toxA PCR for the monitoring of PAR in pigs is substantially improved by a novel quantitative real-time PCR.
Reference strains used in this study
Pasteurella multocida ssp. multocida
Pasteurella multocida ssp. multocida
Pasteurella multocida ssp. multocida
Pasteurella multocida ssp. multocida
Nasal swabs were collected from a total of 203 feeder pigs on farms affected by PAR . Cotton swabs were transported in Amies medium in screw cap plastic vials (VWR, Dietikon, Switzerland) to the lab. Swabs were streaked onto selective blood agar plates according to Rutter et al.  (PB5175A, Oxoid, Switzerland), incubated for 24 h at 37 °C under 5% CO2. From each agar plate Pasteurella-like colonies or if necessary an arbitrarily taken fraction from the bacterial lawn were subjected to DNA extraction.
Genomic DNA was released by heat lysis of bacterial colonies resuspended in water on a thermal shaker for 10 min at 99 °C. After centrifugation at 17,000g for 3 min the supernatant was transferred into a fresh tube and stored at –20 °C until further use. Bacteria from nasal swab cultures were processed accordingly.
Primer and probe sequences with their respective dye and quencher used for the qRT-PCR
ATTO 647 N-AGCACCCAGTCCGCCCTGAGCA-BHQ-3
Gene specificity of both primers and the probe were confirmed by BLAST searches. Primers were synthesized by Microsynth (Balgach, Switzerland). DNA probes were supplied by Eurogentec S.A. (Seraing, Belgium). The probes were quenched by black-hole non-fluorescent quenchers at the 3′-end. Rox dye (Life Technologies, Darmstadt, Germany) was used as an internal reference for normalization and data analyses. An internal amplification control (IAC) was introduced for monitoring each reaction, since bacterial lysates could contain inhibitory substances. Five femtogram (fg) of the pEGFP-1 strandard vector (BD Bioscience Clonetech, USA) was used as IAC template, and a 177 bp amplicon  was amplified with primers and the probe given in Table 2. DNA (1 pg) from a toxA-positive and a-negative P. multocida reference strain (ATCC 12948, ATCC 43137) were used as controls in each qRT-PCR run.
The qRT-PCR was performed on an ABI 7500 Fast Real-Time PCR Instrument (Applied Biosystems) using the Path-ID™ qPCR Master Mix (2×) (Life Technologies). Each reaction contained 5 µl master mix, 200 nM of each eGFP-1-F and eGFP-10-R primer , 1 µl IAC eGFP plasmid DNA (5 fg), 25 nM ATTO 647 N-labeled eGFP probe, 400 nM of each toxA-F and toxA-R primer, 200 nM FAM labeled toxA probe, 2 µl template DNA and nuclease free water to a final reaction volume of 10 µl.
Cycling conditions were 10 min at 95 °C, followed by a two-step cycling stage of 40 cycles of 15 s at 95 °C and 60 s at 60 °C. Data analysis was handled by the 7500 Software version 2.0.4 (Life Technologies). Samples were considered positive when presenting a typical amplification curve with a Ct value of ≤38 for toxA and a Ct value of ≤32 for the IAC. Analyses of samples with IAC Ct values >32 were repeated after reduction of PCR-inhibitory substances by 1:2 and 1:10 dilution.
Determination of the LOD of the qRT-PCR
Genome equivalents P. multocida ATCC 12948
A. toxA (FAM)
B. eGFP (ATTO 647N)
The sensitivity of both the qRT-PCR and the conventional PCR assays was confirmed by testing toxA-positive P. multocida strains. The specificity of the qRT-PCR was confirmed by analysis of 32 different bacterial species (Additional file 1).
An amplification plot of a dilution series of toxA was created (Additional file 2). The efficiency of the qRT-PCR was determined by the use of serial dilution standard curves. The linear correlation coefficient r2 for the toxA gene target was 0.999, showing a high degree of linearity of the qRT-PCR. Based on the slope of the standard curve (–3.348), the amplification efficiency was calculated as 98.9%. At DNA template amounts >1μg, the eGFP amplification was reduced and was completely inhibited with 1.8 μg template DNA. For diagnostic application of the qRT-PCR, a DNA template of maximum 1 μg DNA template was used. To determine the LOD of the assay the acceptance criteria of Ct < 38 and a standard deviation of <0.5 were used. With a LOD of 10 GE, the qRT-PCR showed a tenfold higher sensitivity than the conventional PCR with a LOD of on average 100 GE per PCR reaction . The specificity of both PCR assays was confirmed by repeated test of toxA-positive P. multocida strains. The panel of 32 heterologous bacteria scored negative (Additional file 1). Overall, the qRT-PCR assay revealed a high precision as confirmed by the inter- and intra-assay coefficient of variations (CV) of the Ct values of the positive control as well as the IAC from 52 independent PCR runs. The CV inter-assay values were 5.9% for toxA and 2.7% for IAC; the intra-assay CV values were 1.1% for toxA and 0.58% for IAC.
By comparative analysis of 203 nasal swabs from pigs suspected for PAR, 90 samples (44.3%) were identified as toxA gene-positive by both PCR protocols. A sample of 11 swabs (5.4%) were identified as toxA gene-positive by qRT-PCR (Ct values 16–33.5), whereas the conventional PCR revealed unevaluable results (e.g. several PCR amplifications products of different but unexpected sizes) despite the positive and negative control reactions performed as expected. Overall, the qRT-PCR detected toxA-positive P. multocida in 101 swabs (49.8%). By comparison, the conventional PCR was less sensitive with 90 samples (44.3%) identified as toxA-positive P. multocida.
Noteworthily, a Pasteurella canis strain from the skin wound of a cat was identified as toxA-positive by our novel qRT-PCR. This finding was verified by two independent PCR reactions: Species identification was confirmed by the P. canis-specific PCR of Krol et al. , targeting sodA, a housekeeping gene encoding manganese-dependent superoxide dismutase. In addition to the PCR species identification, strain ZH 401 was confirmed as P. canis by MALDI-TOF analysis. Moreover, a partial fragment of toxA from P. canis ZH 401 has been sequenced and shows a few point mutations in comparison to the highly conserved sequences of toxA-positive P. multocida strains (Additional file 3). The presence of toxA in our feline P. canis was confirmed with an in-house toxA-PCR (primer 34_ToxA 5′ ACTGTAAAAGGAAAAAGTGCCGATG 3′ and 35_ToxA_rev 5′ AAGAGGAGGCATGAAGAGTGC 3′) resulting in a 3792 bp toxA specific amplicon. The expression of toxA in strain ZH 401 was confirmed with the aid of a P. multocida toxin (PMT) ELISA (Oxoid, Pratteln, Switzerland). The finding of toxA in other bacteria than P. multocida is of particular interest. Since toxA is encoded within a lysogenic prophage, it may be transferred to other bacteria (as in our case to P. canis) by transduction .
To conclude, our novel qRT-PCR is highly efficient and robust to diagnose toxA-positive P. multocida in nasal swabs. Compared with the conventional PCR, the qRT-PCR is more sensitive and specific and time saving. This qRT-PCR will facilitate large scale screening to monitor PAR in swine populations.
SS and MMW designed and coordinated the study. DF performed the experiments. SS, DF and MMW participated in authoring the manuscript. All authors read and approved the final manuscript.
We thank Ella Hübschke for the excellent technical assistance in the diagnostic laboratory.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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