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Brief Communication |
Correspondence: 1Corresponding Author: VMD, New Bolton Center, 382 West Street Road, Kennett Square, PA 19348
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Abstract |
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Key Words: Johne's disease • liquid culture • mycobacterium • paratuberculosis • real-time PCR
Paratuberculosis (Johne's disease) is a chronic enteric infection of cattle and other ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Identification of infected animals, which may show no clinical signs, often entails culture of MAP from fecal specimens. Broth culture methods that use automated detection of bacterial CO2 generation or O2 consumption are gaining popularity, because of the shorter culture time necessary to detect MAP growth compared with methods that use solid media. However, once bacterial growth is detected in the broth tube, acid-fast staining, subculturing on solid media, or polymerase chain reaction (PCR) is necessary to confirm the presence of MAP in the sample.
The PCR method represents a rapid and specific means to confirm MAP in broth culture.2,3,5 Previous studies have shown that the concentration of MAP in broth culture tubes is well above the PCR detection threshold at the time the automated detection system signals a positive culture.2,3 In those studies, DNA for MAP PCR testing was harvested by using commercial kits that used proteinase K digestion and column purification. These procedures were presumed necessary for the release of DNA from mycobacterial cells and for separating DNA from PCR inhibitors that potentially could be contained within the culture media. The purpose of the study reported here was to evaluate 2 time-, labor-, and money-saving protocols for harvesting DNA from broth culture for subsequent real-time PCR confirmation of MAP. One method involved direct addition of unprocessed culture broth to the PCR reaction mixture. The second method included immersion of the sample in boiling water for 5 minutes, as an attempt to reduce PCR inhibitors and to effect release of DNA from cells. These methods were compared with a commercially available DNA extraction method that involved mechanical disruption with glass beads and chaotropic purification of DNA. A secondary objective was to determine if PCR could detect MAP growth before signaling "instrument positive," which could potentially shorten the time required to detect positive samples.
In the first experiment, fecal samples from 7 naturally infected cattle and 3 dilutions of pure MAP standard were used for inoculation into the broth media. The 7 fecal samples represented 2 light shedders (84 CFU/g, 92 CFU/g), 2 medium shedders (350 CFU/g, 450 CFU/g), and 3 heavy shedders (>2000 CFU/g) based on results of culture on Herrold's egg-yolk medium. The fecal samples were processed for broth culture by using a standard hexadecylpyridinium chloride decontamination-centrifugation double-incubation method previously described.7 The 3 dilutions of MAP standard were prepared from a frozen stock of a field isolate (ATCC 700535). The final concentrations of MAP in the 3 dilutions were 2600 CFU/ml, 260 CFU/ml, and 26 CFU/ml, respectively. Each broth culture tube was inoculated with 100 µl fecal preparation or MAP standard. In the second experiment, in which the effect of varying egg-yolk volumes was evaluated, 2 of the fecal specimens from the heavy shedding cows and one from the medium shedders were processed as above and were inoculated into the broth culture tubes.
All experiments were conducted by using mycobacterial growth indicator tubes (MGIT) that use a fluorescence indicator to detect bacterial oxygen consumption.a The tubes contain 7 ml of a modified Middlebrooks 7H9 broth, to which was added 800 µl of oleic acid/bovine albumin/catalase/casein supplement,b and 30 µl of amphotericin B /nalidixic acid/vancomycin mixture (5 mg/ml of each). In the first experiment, 500 µl of 50% egg-yolk emulsionc was added to each MGIT tube, and, in the second experiment, the effect of various egg-yolk quantities was evaluated by amending MGIT tubes with either 250 µl, 500 µl, or 1000 µl of egg-yolk emulsion. Culture tubes were inoculated with 100 µl of fecal preparation or MAP standard dilution and were incubated in the automated detection system instrument,d until signaled "positive" based on an algorithm by using measurements of fluorescence.
At the conclusion of the incubation period, culture tubes were vortexed and divided into 3 aliquots. For the "direct" method, 2.5 µl of culture medium was added directly to the PCR mixture, without processing. For the "boil" method, 1.0 ml of culture medium was placed in a microcentrifuge tube and was immersed in boiling water for 5 minutes, after which 2.5 µl of the boiled medium was added to the PCR mixture. The third aliquot was processed by using a commercially available DNA extraction procedure that was optimized for MAP quantitative real time PCR (qrtPCR) on fecal samples.e Manufacturer's recommendations were followed, with the exception that 1.0 ml of culture medium was the starting substrate rather than 1.0 g of feces. Briefly, the extraction procedure involves bead-beating the sample, followed by chaotropic purification. After elution from the purification column, 2.5 µl DNA solution was added to the PCR reaction mixture.
A commercially available qrtPCR procedure that targets the MAP hspX gene was used to amplify DNAe by using a real-time PCR thermal cyclerf with probe-based 6-carboxyfluorescein dye detection. The reaction volume was 25 µl, and after a 95°C hold for 10 seconds, the samples were subjected to 50 cycles of 95°C for 5 seconds and 62°C for 30 seconds. Each sample was run in duplicate, and the number of cycles to reach threshold was recorded for each sample. The number of cycles to threshold correlates with the amount of DNA template in the original sample, with each log dilution of MAP DNA, resulting in 3.3 additional cycles to threshold (data not shown).
In the first experiment, the 7 fecal specimens and the 3 MAP standard dilutions were each inoculated into 3 MGIT tubes. For each specimen, 1 MGIT tube was processed by all 3 DNA harvesting methods and was subjected to qrtPCR on day 0 (i.e., no incubation), 1 tube was processed after 8 days of incubation, and the third tube was processed on the day signaled instrument positive. In the second experiment, each of the 3 fecal preparations was inoculated into MGIT tubes that contained the 3 different volumes of egg-yolk emulsion additive. Tubes were processed by all 3 DNA harvesting methods and were subjected to qrtPCR after 8 days and 11 days of incubation, and on the day signaled instrument positive.
Analysis of variance was used to compare the mean number of PCR cycles to positive threshold (Ct) for the 3 DNA harvesting methods at the standard egg-yolk volume (500 µl) for the samples collected at time to instrument signal culture positive. The differences in Ct for samples incubated with different egg-yolk volume were also compared for each DNA harvesting method by using analysis of variance. The null hypothesis was rejected when P < 0.05. For the purposes of statistical analysis, a value of 51 cycles was assigned to those specimens that were still negative at the conclusion of the 50-cycle qrtPCR run.
In the first experiment, with constant egg-yolk volume (500 µl), all the samples signaled instrument culture positive between 10 and 15 days for the heavy shedders, up to 31 days for the light shedders. When tested on day 0 or 8 of incubation (i.e., before signaling instrument positive), none of the 10 samples were positive on qrtPCR, regardless of the DNA harvesting method used. The results for the 3 DNA extraction methods on samples processed at time to signaling instrument positive are shown in Table 1. One sample harvested with the boil method did not test qrtPCR positive, and 4 were negative when the direct method was used, compared with 100% detection when the commercial DNA harvesting procedure was used. The sample that was negative by the boil method was the middle dilution of MAP standard (260 CFU/ml starting concentration); all 7 fecal samples were positive. The number of cycles to threshold positive, which is, in turn, an indication of the concentration of DNA in the sample, was significantly different for the 3 methods (Table 1).
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Regardless of the starting quantity of MAP, the number of cycles to threshold positive between samples had minimal variation (within each particular extraction method). This would be expected, because each sample was incubated until signaled instrument positive, and, presumably, the samples all signaled positive when MAP concentration in the sample reached a similar concentration. Those samples with less MAP in the starting inoculum required a longer incubation to instrument signal positive.
The samples extracted by the commercial method had lower values for qrtPCR Ct compared with the boil method and the direct method, suggesting that those samples had a greater quantity of target DNA. This would be expected, as with the commercial method, the amount of DNA in 1.0 ml is concentrated to a volume of 50 µl. Therefore, the 2.5 µl DNA template added to the qrtPCR reaction represents a 20x concentration compared with the boil and direct methods, in which the 2.5 µl DNA template is not concentrated. Alternatively, it is possible that inhibitors of the PCR reaction, which might reduce the efficiency and thus prolong the number of cycles to reach positive threshold, could be removed by the more elaborate commercial methods but not by boiling. This is supported by the deleterious effect of increased egg-yolk volume on qrtPCR results. Presumably, the effect of egg-yolk volume was greatest in the samples harvested when using the direct method, because this method does not abrogate the effects of egg yolk in the sample, whereas boiling or chaotropic purification does.
Although most previous studies used commercial DNA extraction kits with proteinase K digestion,1–3,5 1 study reported successful qrtPCR confirmation after heating to 99°C.6 However, in that study, centrifugation and washing of the pellet to concentrate MAP and to remove inhibitors was performed before heating the sample.
The results of this study regarding successful detection of MAP DNA from samples at time to instrument signal positive are similar to previous reports, which suggested that MAP concentration was well above the qrtPCR detection threshold.2,3 However, the studies reported here suggest that attempts to significantly shorten the culture time by testing before instrument signaling positive will not be fruitful. Based on the reported 1- to 3-day generation times of MAP in log phase growth,3,4 shortening the incubation time by several days could result in several orders of magnitude less MAP concentration in the broth (as much as 10x for each 3.3 days), which results in MAP concentrations below the qrtPCR detection threshold. Although not evaluated in this study, it may be possible to identify a time before instrument signal positive (but longer than the incubation of 8 or 11 days examined here) at which time PCR confirmation would be successful.
In summary, the boil method of DNA harvesting represents an inexpensive and time- and labor-saving method to prepare DNA from broth cultures for qrtPCR confirmation of MAP. Those few samples from instrument signal positive culture tubes that give negative qrtPCR results with the boil method could be re-extracted with a commercial method to confirm the negative status. This method should not be applied before the time that the sample is signaled culture positive by the instrument, because false-negative results could result.
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Acknowledgments |
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Sources and manufacturers |
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TOPSources and manufacturersAbstractReferences |
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a. Bactec MGIT ParaTB Medium tube, BD Diagnostic Systems, Sparks, MD.
b. Bactec MGIT ParaTB Supplement, BD Diagnostic Systems, Sparks, MD.
c. Difco 50% Egg Yolk Enhancement, BD Diagnostic Systems, Sparks, MD.
d. Bactec MGIT 960 Mycobacterial Detection System, BD Diagnostic Systems, Sparks, MD.
e. VetAlert, Tetracore, Inc. Rockville, MD.
f. Smart Cycler, Cepheid, Sunnyvale, CA.
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