Detection of Mycoplasma hyopneumoniae by Air Sampling with a Nested PCR Assay

Abstract

This article describes the first successful detection of airborneMycoplasma hyopneumoniae under experimental and field conditions with a new nested PCR assay. Air was sampled with polyethersulfone membranes (pore size, 0.2 μm) mounted in filter holders. Filters were processed by dissolution and direct extraction of DNA for PCR analysis. For the PCR, two nested pairs of oligonucleotide primers were designed by using an M. hyopneumoniae-specific DNA sequence of a repeated gene segment. A nested PCR assay was developed and used to analyze samples collected in eight pig houses where respiratory problems had been common. Air was also sampled from a mycoplasma-free herd. The nested PCR was highly specific and 104 times as sensitive as a one-step PCR. Under field conditions, the sampling system was able to detect airborne M. hyopneumoniae on 80% of farms where acute respiratory disease was present. No airborne M. hyopneumoniae was detected on infected farms without acute cases. The chance of successful detection was increased if air was sampled at several locations within a room and at a lower air humidity.

Incorporating polymerase chain reaction-based identification, population characterization, and quantification of microorganisms into aerosol science: A review

“…The process of nesting almost always decreases the detection level. Stark et al (1998) observed a 10 4 increase in sensitivity by using nested primers, although the increase is usually approximately 1-2 orders of magnitude.…”

“…Samples on solid supports were eluted with 0.1% Triton X-100 solution and ground in liquid nitrogen to lyse spores. DNA was purified by phenol:chloroform extraction and DNA concentrated and cleaned by a spin column Aerosols sampled from pig rooms on seven commercial farms, Mycoplasma hyponeumoniae, nested PCR increased sensitivity by 10 4 times (Stark et al, 1998) 0.2 mm polyethersulfone filters at 8.3-20 L min À1 Filters were dissolved in chloroform, shaken, and then DNA was extracted from the solution by phenol:chloroform extraction, followed by ethanol precipitation and centrifugation of DNA Outdoor wastewater treatment plant, chemical plant, and indoor office building, Legionella spp., PCR more sensitive than plate counts (Pascual et al, 2001) Impacted onto a petri dish containing 20 mL of phosphate buffered saline, flow at 100 L min À1 Cells concentrated by centrifugation and lysed by freeze-thaw process. No purification Indoor and outdoor urban aerosols, Pneumocystis carinii, limit of detection was 10 2 cells without inhibition (Maher et al, 2001) 0.45 mm polyvinylidene difluoride filters at 0.4 L min À1 for 24 h Cells were lysed directly on the filter using extraction buffer and DNA was purified by phenol:chloroform followed by ethanol precipitation.…”

“…Filter materials that have been successfully used for cell concentration and elution in aerosol PCRbased studies include nucleopore polycarbonate filters (Paez-Rubio et al, 2005;Palmgren et al, 1986), PTFE (Bej et al, 1991;Myatt et al, 2004), polyethersulfone (Angenent et al, 2005;Stark et al, 1998), and mixed cellulose ester filters (Alvarez et al, 1995) (Table 2). Polycarbonate is the most common type and has been used in greater than 50% of previous PCR-based aerosol studies.…”

“…Depending on the primers used, analysis can be extended toward microorganism identification, population analysis, or quantification. The most common analysis in aerosol studies has involved identifying a specific pathogenic virus or species of bacteria or fungi Maher et al, 2001;Mastorides et al, 1997;Myatt et al, 2004;Pascual et al, 2001;Sawyer et al, 1994;Stark et al, 1998;Wakefield, 1996;Wan et al, 2004). For these types of studies, single agents can be chosen and their DNA amplified from an environmental sample by using a specific set of primers.…”

Relationship between airborne detection of influenza A virus and the number of infected pigs

“…Airborne spread of swine pathogens has been a topic of discussion in the last decades. There are reports on the detection of swine pathogens (bacteria and virus) in air samples (Bourgueil et al, 1992;Torremorell et al, 1997;Stark et al, 1998;Hermann et al, 2008;Weesendorp et al, 2008;Dee et al, 2009;Verreault et al, 2010). In addition, studies conducted in pigs to understand the regional spread of porcine reproductive and respiratory syndrome virus (PRRSv) and Mycoplasma hyopneumoniae confirmed that viable pathogens could travel between 3.5 and 9.1 km from the presumed source of infection Otake et al, 2010).…”