Efficient, Validated Method for Detection of Mycobacterial Growth in Liquid Culture Media by Use of Bead Beating, Magnetic-Particle-Based Nucleic Acid Isolation, and Quantitative PCR

Abstract

Pathogenic mycobacteria are difficult to culture, requiring specialized media and a long incubation time, and have complex and exceedingly robust cell walls. Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease, a chronic wasting disease of ruminants, is a typical example. Culture of MAP from the feces and intestinal tissues is a commonly used test for confirmation of infection. Liquid medium offers greater sensitivity than solid medium for detection of MAP; however, support for the BD Bactec 460 system commonly used for this purpose has been discontinued. We previously developed a new liquid culture medium, M7H9C, to replace it, with confirmation of growth reliant on PCR. Here, we report an efficient DNA isolation and quantitative PCR methodology for the specific detection and confirmation of MAP growth in liquid culture media containing egg yolk. The analytical sensitivity was at least 10 4 -fold higher than a commonly used method involving ethanol precipitation of DNA and conventional PCR; this may be partly due to the addition of a bead-beating step to manually disrupt the cell wall of the mycobacteria. The limit of detection, determined using pure cultures of two different MAP strains, was 100 to 1,000 MAP organisms/ml. The diagnostic accuracy was confirmed using a panel of cattle fecal (n ‫؍‬ 54) and sheep fecal and tissue (n ‫؍‬ 90) culture samples. This technique is directly relevant for diagnostic laboratories that perform MAP cultures but may also be applicable to the detection of other species, including M. avium and M. tuberculosis. P aratuberculosis, or Johne's disease, affects many economically important livestock species, including cattle, sheep, goats, and deer (1). It is caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is characterized by chronic enteritis, wasting, and death. Control programs attempting to limit the impact and spread of the disease in many countries are based on the detection of infected animals by using fecal culture, which is one of the most specific antemortem tests for the diagnosis of paratuberculosis infection (2). The gold standard indications for disease confirmation are a pathological lesion consistent with paratuberculosis and a positive culture for MAP from the affected tissues (3).For many mycobacterial species, culture requires long incubation times, from weeks to months, and highly specialized media. For MAP, liquid culture media, such as Bactec 12B medium, have been found to be more sensitive than solid culture media, such as Herrold's egg yolk medium (HEYM) supplemented with mycobactin J (4-6). MAP in cultures with positive growth is confirmed by the demonstration of mycobactin dependency or directly by molecular techniques (7). The advantages of liquid culture media systems include shorter culture incubation times (5, 7), and, importantly, some liquid culture media are capable of supporting the growth of all strains of MAP, including the fastidious sheep strain (8). Monitoring of bacterial growth in liquid cul...

Improved Culture Medium (TiKa) for Mycobacterium avium Subspecies Paratuberculosis (MAP) Matches qPCR Sensitivity and Reveals Significant Proportions of Non-viable MAP in Lymphoid Tissue of Vaccinated MAP Challenged Animals

“…Previous comparative studies of qPCR and conventional culture have demonstrated good correlation in high sample loads present in feces (Douarre et al, 2010; Mita et al, 2016) but the inability of conventional culture methods to accurately culture low loads of viable organisms from clinical samples introduces problems. Culture sensitivity for MAP has only ever been as good as 2–3 log 10 (Ricchi et al, 2016), thus at low loads correlations to DNA presence are difficult to obtain and the true relationship between genome equivalent values and the demonstrable viable MAP count in these samples remains uncertain (Kralik et al, 2012; Plain et al, 2015). The underlying reasons for these discrepancies are probably multi-variant.…”

A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays

“…paratuberculosis cells, and the DNA extraction/qPCR detection method (27). The linearity of the growth curve confirms that the growth rate in relation to the starting inoculum concentration is relatively constant for M. avium subsp.…”

“…An aliquot from each of the serial dilutions was collected at the time of culture inoculation (T 0 ) and then stored at Ϫ80°C, and subsequent aliquots from each serial dilution were taken weekly for 3 weeks and stored at Ϫ80°C. The frozen aliquots were then thawed and subjected to DNA extraction and IS900 qPCR using a high-throughput 96-well-plate DNA extraction method, which included the magnetic bead-based DNA isolation method and IS900 qPCR, as previously validated and detailed above (27). This viability curve was repeated 20 times over 5 different experiments to ensure the reliability of the results.…”

“…DNA was isolated, and quantification of M. avium subsp. paratuberculosis DNA from the suspensions using an IS900 qPCR assay was done as previously described and as detailed below (27). The entire experiment was repeated twice to examine the reproducibility and consistency of the results.…”

“…Detection of live M. avium subsp. paratuberculosis cells was performed by using a modification of the M7H9C liquid culture method described previously (10,27). Briefly, multiple 10-fold dilution series of M. avium subsp.…”