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

Abstract

Actinobacillus pleuropneumoniae (APP), the causative agent of porcine pleuropneumonia, is highly contagious and responsible for high morbidity, mortality, and economic losses in the swine industry worldwide, but quick serotyping and diagnosis are still not widely available. In this study, we sought to validate the use of Whatman FTA® cards for collection and processing of A. pleuropneumoniae isolates, or porcine lung tissue samples, for direct use in diagnostic multiplex PCRs. We have optimized the processing of 3-mm discs punched from FTA® cards loaded with cultured A. pleuropneumoniae, or imprinted on lesioned regions of lung tissue, with only three distilled water washes before addition into our APP-multiplex PCR (mPCR) assay for rapid, low-cost identification and serotyping. DNA captured on FTA® cards generated the same diagnostic PCR results as DNA extracted using commercial kits for 85 A. pleuropneumoniae clinical isolate cultures and 22 lung samples. Additionally, bacterial DNA bound to FTA® cards was detectable by PCR after 6 months of storage at 37°C. This study provides simple, efficient, rapid, and practical sample processing for detection and molecular serotyping of A. pleuropneumoniae.

JMM Profile: Actinobacillus pleuropneumoniae: a major cause of lung disease in pigs but difficult to control and eradicate

“…Lung samples from acute lesions are preferred, since those from chronic cases or lesions at the abattoir can be negative for culture [ 3 ]. A simple rapid protocol describing identification (through the apxIV gene) and serotyping by multiplex PCRs from imprinted lung lesion material on FTA cards has recently been published [ 7 ].…”

Rapid Detection of Actinobacillus pleuropneumoniae From Clinical Samples Using Recombinase Polymerase Amplification

“…RPA reagent concentrations remained as above, however reaction volumes were increased to 25 µL to account for the FTA card disc. Samples on FTA classic cards (Whatman plc, Little Chalfont, Buckinghamshire, UK, Cat #WB120205) were prepared by pressing the card surface against lung tissue as previously described (23). FTA cards were allowed to dry fully prior to being processed, 3 mm discs were removed from the inoculated card with a sterile biopsy punch (Integra Miltex Cat #12-460-406, Fisher Scientific, Loughborough, UK), which was rinsed in ethanol and deionized water between uses to prevent crosscontamination.…”

“…To evaluate the use of lung smears made on FTA cards as a sample isolation method amenable to rapid a diagnostic test for APP, a subset of frozen lung samples (n = 13) were inoculated onto FTA cards, which were then dried and washed using our previously described water-wash protocol validated for use in PCR (23). In the clinical evaluation above, one of the lungs were scored as having been negative for APP, three of the lungs were determined to contain low, four medium and five high bacterial loads by qPCR.…”

“…We have previously described an alternative sampling method, directly imprinting infected tissue on FTA cards, which chemically lyse and entrap nucleic acids. We have shown the effectiveness of this method, when combined with a simple water wash and our multiplex PCR, for the detection and serotyping of APP (23). Further adaptation of this sample preparation for combination with RPA could prove a powerful tool to screen for APP infection on-site, i.e., directly at the farm or abattoir.…”

Novel DNA Markers for Identification of Actinobacillus pleuropneumoniae

“…DNA-based detection methods such as polymerase chain reaction (PCR) can be highly specific, allowing discrimination of different species when the targeted DNA sequences are sufficiently unique. Amplification of A. pleuropneumoniae - specific DNA in pig-derived samples (e.g., lung tissues, nasal swabs, tonsils, and oral fluids) is therefore exploited for disease diagnosis ( 9 – 12 ).…”