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A testing scheme for the detection of Mycobacterium avium subsp. Paratuberculosis in bovine feces utilizing the ESP Para-JEM liquid culture system

Correspondence: ¹Corresponding Author: Sreekumari Rajeev, University of Georgia, Veterinary Diagnostic and Investigational Laboratory, 43 Brighton Road, Tifton, GA

	Abstract

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A testing scheme for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in broth cultures of bovine fecal samples carried out in ESP para-JEM System was evaluated. The scheme included acid-fast staining (on signal-positive and signal-negative samples), and confirmation by PCR for 2 MAP-specific targets and subculture of all acid-fast positive PCR-negative samples. Two hundred and fifty bovine fecal samples were evaluated for the presence of MAP using this scheme. Thirty-seven (15%) of 250 fecal samples had a positive culture result when the proposed testing scheme was used, compared to 14 (6%) positive results when using the standard ESP para-JEM protocol (requiring samples to have a positive signal from the system, a positive acid-fast stain, and a positive IS900 PCR result), and 20 (8%) positives when conventional culture was performed on Herrold egg yolk (HEY) media. A preliminary comparison of real-time and conventional PCR on DNA extracted from 15 MAP-positive broth cultures by 3 different protocols suggested that conventional PCR may be a better choice for the confirmation of the presence of MAP in the liquid cultures than real-time PCR.

Key Words: ESP • Johne disease • paratuberculosis • PCR

	Introduction

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Johne disease (JD) or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is an economically important disease affecting a wide variety of animal species.7 Rapid and accurate diagnosis of MAP infection is critical for JD control programs. Organism detection methods and measurement of host immune response to infection are the most commonly used methods for JD diagnosis.7,12,16 Although assessment of host immune response can be utilized to identify infected herds, these assays are not well suited to identify clinically normal, infected animals within a herd.2,4 Fecal culture has merit over immunological methods because of the higher specificity and sensitivity. Hence, it is the method of choice for the detection and removal of individual infected animals from a herd. Outcome of fecal culture is influenced by the infection stage of the animal, sample processing and decontamination procedures, the culture method used, and the degree and nature of contamination in fecal samples.12,16 Fecal culture using Herrold egg yolk (HEY) media is the most commonly used culture method for the detection of MAP and because of MAP's inert biochemical profile, its dependency on the siderophore, mycobactin J, is utilized for confirmation.7

Automated liquid culture systems coupled with molecular detection offer a faster turn-around time with improved sensitivity and specificity for the detection of mycobacteria in clinical samples.6,10,18,19 After sample processing and inoculation into the broth media, no further operator input is required until the system signals positive samples, whereas conventional culture requires manual reading of inoculated HEY tubes at 2–4-week intervals. Another important advantage of using the liquid culture system is a more rapid turn-around time in identifying infected animals. The test results can be finalized in 6 weeks with the broth system instead of 16 weeks in conventional HEY cultures.10,14 Because of the possible presence of other non-MAP mycobacterial species in bovine fecal samples, acid-fast staining alone is not sufficient for MAP confirmation in broth cultures. PCR using IS900 is one of the most common molecular methods used for confirmation in conjunction with the liquid culture system, and several IS900 PCR protocols have been published.5,7,17 Since specificity of IS900 was a concern,3,13 we previously identified and evaluated a real-time PCR protocol based on the MAP-specific genomic target 251.14 In the present study, a testing scheme was evaluated that combined broth culture, acid-fast staining, PCR (targeting IS900 and 251), and subculture of inconclusive samples for the detection and confirmation of MAP from broth cultures.

	Materials and Methods

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Culture
Two hundred and fifty bovine fecal samples submitted to the Ohio Animal Disease Diagnostic Laboratory (ADDL) for routine MAP culture were tested in this study. Fecal samples were processed and inoculated into ESP para-JEM broth^a according to the manufacturer's protocol with modifications. Briefly, 2 g of feces were mixed with 35 ml of sterile distilled water and allowed to settle for 30 min. Five milliliters of the supernatant was added to 25 ml of 0.9% HPC (hexadecylpyridinium chloride monohydrate) solution in sterile distilled water. After overnight incubation, the sample was centrifuged at 900 x g for 20 min. The pellet was resuspended in 1 ml of antibiotics (ESP para-JEM AS^a) according to manufacturer's instructions and incubated for 18–24 h. Two sets of fecal samples from the same animal were processed as described above and samples were combined and mixed thoroughly after the treatment with antibiotic mixture to meet the volume requirement and to assure uniformity in the final sample that was inoculated into HEY tubes and ESP broth. One milliliter of the sample was inoculated to ESP para-JEM culture bottles containing supplements, para-JEM EYS,^a para-JEM AS,^a para-JEM GS,^a and para-JEM blue^a as per manufacturer's instructions. One hundred and fifty microliters of the each sample were also inoculated to each of 3 HEY tubes^b with mycobactin J and 1 tube without mycobactin J.^b The HEY tubes were incubated in an aerobic incubator at 35°C for 16 wks and were examined every 2 wks for the presence of MAP colonies. The ESP bottles were incubated in ESP culture system II^a until the system showed positive signals or up to 42 d, whichever came first. The signal-positive bottles were removed from the system and evaluated by acid-fast staining and acid-fast bacilli (AFB)-positive samples were confirmed by PCR. The broth samples with no AFB present were further incubated in the system. At the end of 42 days all of the remaining samples were acid-fast stained, and samples with AFB were subjected to PCR. All the AFB-negative samples were also subjected to PCR. All signal negative and signal positive broth samples were subcultured into one HEY tube with mycobactin J, one HEY tube without mycobactin J, and a blood agar plate to evaluate the presence of other viable bacteria.

Acid-fast Staining of Broth Cultures
The ESP broth culture bottle was shaken in an IKA VIBRAX VXR basic Shaker^c for 5 min at 2000 rpm, and 25 µl of the sample was placed on a clean microscopic slide. The dried smear was heat fixed and stained by a fluorochrome acid-fast staining method.8 Briefly, smears were flooded with auramine O/ rhodamine fluorescent stain for 3–5 min. The smears were decolorized with fluorescent decolorizer for 2 min after washing with deionized water. Slides were counterstained with potassium permanganate for 2 min. After washing and drying, slides were examined in a Nikon Eclipse E400 microscope with a fluorescent attachment using a filter with excitation at 450–490 nm and a 40x objective.

Dna Extraction and Pcr
After shaking the broth culture for 10–15 min as described above, 1 ml of the culture was removed and centrifuged at 400 x g for 1 min in an Eppendorf microcentrifuge. This was done to remove egg yolk and particulate debris. The supernatant was transferred to a new tube and was centrifuged at 16,000 x g for 3 min. The pellet was washed 4 times with 800 µl of 10 mM Tris-HCL at 16,000 x g for 3 min. Fifty microliters of Lyse-N-Go PCR reagent^d was added to the pellet and incubated at 95°C for 1 h. This sample was centrifuged at 16,000 x g for 1 min and the supernatant was harvested for DNA extraction using QIAamp DNA Mini kit^e according to manufacturer's protocol. Real-time PCR was performed on these samples targeting IS900 as previously described.14 IS900 real-time PCR positive samples were further tested using 251 real-time PCR. Acid-fast positive, IS900-negative broth samples were subcultured into HEY tubes with and without mycobactin J (100 µl inoculum) and to new bottles of ESP broth (500 µl inoculum) and incubated as described in the primary culture. Samples that yielded IS900-positive, 251-negative results were also subcultured similarly. After 4 wk, HEY tubes were examined for colonies demonstrating mycobactin J dependency and the broth cultures were tested by AFB staining and by IS900 and 251 real-time PCR.

Comparison of Real-time and Conventional Pcr
A pilot study was conducted to evaluate the efficiency of real-time and conventional PCR and 3 different DNA extraction protocols to confirm MAP from AFB-positive broth samples. The DNA from 15 MAP confirmed and 2 negative broth samples was extracted using 3 different protocols. In the first protocol, 1 ml of broth culture was subjected to a single-step lysis and extraction using Lyse N Go PCR reagent^d and the supernatant was used for PCR. In the second protocol the supernatant from protocol 1 was further extracted using QIAamp DNA Mini kit.^e This protocol was performed to obtain more purified DNA. In the third protocol, DNA was extracted from 1 ml of broth sample using QIAamp DNA Mini kit^e using manufacturer's protocol. Conventional and real-time PCRs were performed targeting both IS900 and 251 on all the samples.

Statistical Methods
McNemar's chi-square was used to compare the proportions of positive results obtained in paired testing of samples by 2 different cultures or DNA extraction methods, while Cochran's Q was used for simultaneously comparing 3 or more methods. Exact binomial P-values were reported for McNemar's test, and asymptotic P-values were reported for Cochran's Q. All testing assumed a two-sided alternative hypothesis, and P-values <0.05 were considered statistically significant. Calculations were performed using commercially available statistical software (SPSS version 12.0).

	Results

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Culture and Confirmation
Thirty-seven (15%) of 250 fecal samples had a positive culture result when the proposed ESP testing scheme was used, compared to 14 (6%) positive results when using the standard ESP protocol (requiring samples to have a positive signal from the system's oxygen sensor, a positive acid-fast stain, and a positive IS900 PCR result), and 20 (8%) positives when conventional culture was performed on solid HEY media. The proportions of positive culture results did not differ significantly between the standard ESP protocol and HEY culture (McNemar's chi-square, 1 df, P = 0.109), although both of these methods yielded significantly fewer positives than the proposed ESP testing scheme (Cochran's Q, 2 df, P < 0.001). Figure 1 represents the testing scheme and number of positive and negative samples at each step.

View larger version (19K): [in this window] [in a new window]   Figure 1 Testing scheme used for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples. n = number of samples in each step. * Subculture is done in both ESP broth and HEY tubes. MAP growth in ESP broth was confirmed by PCR and HEY tubes was confirmed by mycobactin dependency. ** This sample was identified to contain Mycobacterium chelonae. *** These samples remain unidentified.

Comparison of Pcr Methods
DNA was extracted from 15 MAP-confirmed broth cultures and 2 MAP-negative broth cultures using 3 different protocols, and conventional PCR and real-time PCR targeting IS900 and 251 was performed. Results of the individual samples are given in Table 1. Conventional PCR for both the IS900 and 251 sequences correctly identified the status of all test samples (15 known to contain MAP and 2 negative controls) regardless of which DNA extraction method was used, but the results of real-time PCR testing were dependent on both the target sequence and the extraction method. In testing for the IS900 sequence, real-time PCR of samples that were processed using the Lyse N Go reagent yielded 2 (12%) positive test results, compared to 11 (65%) positives following the Qiagen extraction, and 13 (76%) positives when a combination of the Lyse N Go and Qiagen extractions was used. There was no significant difference between the proportions of positive results for the two methods that incorporated the Qiagen extraction (McNemar's chi-square, 1 df, P = 0.687), but when the Lyse N Go reagent was used alone there were significantly fewer positives (Cochran's Q, 2 df, P = 0.001).

	Discussion

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Unambiguous detection of MAP is essential for implementation of JD control programs. Therefore multiple steps including acid-fast staining and PCR targeting more than one MAP-specific sequence, are ideal to confirm signal positive samples from an automated broth culture system. ESP culture when used in combination with the developed testing scheme detected 85% more positive samples compared to HEY. The use of the testing scheme developed in this study improved MAP detection. Initial screening of all signal-positive samples from the ESP system by acid-fast staining serves as a visual check point for downstream screening procedures. Severely contaminated samples, which may give a continuous positive signal, can be excluded from downstream procedures. Because 67% of the MAP-positive broth cultures had contaminant bacteria or fungi when subcultured on blood agar, it may be logical to assume that MAP may grow in the broth culture in the presence of contaminating bacteria or fungus. These broth cultures containing AFB can be confirmed by PCR. However, in HEY cultures, the surface of the media may be overgrown with contaminants and it is almost impossible to read contaminated tubes.

A study that compared the results of BACTEC MGIT 960 and ESP Culture System II for the recovery of Mycobacterium spp. from human samples reported a contamination rate of 17.1 and 18.9% respectively.20 This study also included and recommended the inclusion of one solid media and terminal subculture of negative liquid culture medium to obtain a sensitive and accurate detection.20 Terminal subculture of all the signal-negative media is labor intensive, costly, and time consuming. Acid-fast staining of the terminal broth followed by confirmation by PCR or subculture of AFB-positive samples can be an alternative to this. To our knowledge there are no published studies comparing BACTEC, MGIT 960, and ESP Culture systems for the recovery of MAP from fecal samples.

Acid-fast staining using auramine O/rhodamine is more sensitive than conventional acid fast staining such as the Kinyoun method8 and, screening of samples under a 40x objective is sufficient for identifying AFB, whereas, a more tedious examination of slides under an oil immersion objective is required for conventional acid-fast staining when using the Kinyoun method. Highly sensitive IS900 PCR can be used to screen all AFB-positive broth samples and all IS900-positive samples can be further confirmed with another MAP-specific target 251, thereby increasing the specificity of MAP detection. An optimized multiplex PCR targeting both sequences could reduce the cost of testing. Subculture and confirmation of all AFB-positive, but PCR-negative samples is needed to eliminate any false-negative results due to PCR inhibition. It is important to note that 73% of the true positive samples were not PCR positive by the initial screening using real-time PCR. A recently published review presents valuable information about the technical issues that can create false-positive and false-negative results when using an analytically sensitive technique such as PCR that can theoretically detect a single organism in a clinical sample.1 Most PCR assays have very high specificity, but variable sensitivity depending on the type of clinical samples and the DNA extraction protocols used.15 A previous study comparing different DNA extraction methods reflects the difficulty in comparing the experimentally determined detection limits, achieved by spiking the experimental samples, to the true sensitivity of finding bacterial DNA in clinical samples.15 Difficulty in detecting gram-positive bacterial species and mycobacteria by PCR is also associated with problems in breaking the tough cell walls and releasing DNA for amplification.15 For these reasons many PCR diagnostic tests have been given promising analytical results, but are less effective in routine clinical use. These factors must be considered when interpreting the PCR results and appropriate quality control and check points have to be incorporated in the testing protocol. In a previous study, we evaluated an internal control DNA for PCR inhibition and our experience has revealed target dependant PCR inhibition does occur.14 We have observed MAP positive samples that gave positive results with the internal control, but negative results with one or more target sequence. Therefore, incorporation of an internal control may not always be successful in predicting the sensitivity of PCR reaction since the mode of action of inhibitors present in clinical samples could be different. A true signal-positive sample will have 1 x 10⁵ or more organisms per milliliter of the sample.9,10 Therefore acid-fast staining can serve as an excellent check point and is a key step in improving the detection sensitivity.

A recent study demonstrated increased sensitivity of real-time PCR over conventional PCR by using standard dilutions of genomic DNA equivalent and the study suggested that the diagnostic sensitivity of conventional and real-time PCR on ESP broth cultures are equivalent due to the presence of large numbers of bacteria at the time of signal.9 However, this assumption should be interpreted with caution since the number of organisms present in a clinical sample is not the only factor influencing a PCR result.1 Inhibitory components present in fecal material added to broth media may have a negative influence on PCR sensitivity.1 The real-time PCR we used in this study was initially evaluated in 210 MAP field isolates14 and was used routinely for the confirmation of field isolates from HEY tubes. Assuming that PCR inhibitors present in the sample and the PCR technique (real-time PCR vs conventional PCR) itself may contribute false negative results, we compared the results of real-time and conventional PCR targeting both IS900 and 251. Although we used only a limited number of samples in this study, it is important to note the wide variation in results of real-time PCR. Considering the fact that there is no standardized protocol for MAP culture in approved laboratories, this variation in results may have impact on detection and confirmation of MAP.

We also compared the effect of different DNA extraction protocols on PCR results for a limited number of samples. In this work, conventional PCR detected more positive samples compared to real-time PCR regardless of the DNA extraction procedure. Although we do not have an explanation for this difference at this time, it may be logical to assume that because real-time PCR is more complex due to probe binding and hydrolysis steps, and these steps might have been affected by inhibitors. This is supported by the observation that all of the real-time PCR products (from 15 samples we used to compare the results of real-time and conventional PCR with different DNA extraction protocols) had a visible DNA band of correct size on gel electrophoresis. If the PCR reaction was inhibited, a product would not have been visible. Probe binding and hydrolysis are the mechanisms used for fluorescent detection in our real-time PCR reaction. However, when these steps are inhibited, amplification will still take place but there will not be a recordable difference in fluorescent intensity thus resulting in a negative cycle threshold value. As a result of these observations, conventional PCR is now routinely used for confirmation of MAP from broth samples at the ADDL.

Our results also indicate that a selective extraction/target dependant effect of inhibitors may be taking place in the reactions. The use of the QIAamp DNA Mini kit^e by itself or a combination of Lyse N Go PCR reagent and the QIAamp DNA Mini kit^e resulted in more positive results with IS900 real-time PCR. In contrast extraction using only Lyse N go PCR reagent resulted in greater numbers of positive samples with 251 real-time PCR. This observation suggests that copy number of the gene, or the number of organisms present, are not the only factors influencing the sensitivity of PCR. Target-related PCR inhibition is the most likely factor influencing the outcome of the results and this finding needs more investigation. Addition of compounds such as bovine serum albumin to PCR reaction mixture has been reported to alleviate PCR inhibition.11 We have observed that addition of bovine serum albumin to alleviate PCR inhibition has had a positive impact on 251 PCR results but not on IS900 PCR results (data not shown).

Although broth culture systems are more expensive than conventional HEY cultures these automated systems are useful in laboratories handling large numbers of samples since the automated systems reduce the labor involved in manual reading of the tubes. However, our results suggest that the occurrence of false-positive signals may be common in the ESP culture system, possibly due to bacterial or fungal contamination or to unknown mechanical factors interfering with the signaling system. Considering the false-positive signals from the system and false-negative PCR results, we believe that acid-fast staining should be included in the testing scheme. In our approach, the signal-positive broth bottles, which are AFB negative, are reincubated in the system. In the case of broth bottles with turbid media and color change (these bottles will give a continuous signal in the system) indicating contamination, the fecal sample has to be recultured. Using the testing scheme described here laboratories that handle a small number of samples could possibly use the broth culture media without the automated system to eliminate the cost involved in buying the expensive equipment. Acid-fast staining at weekly intervals, or at the end of the incubation period (42 days) followed by the testing scheme described in this study will detect and confirm positive samples. It should be noted that the testing scheme described here has been evaluated only for bovine fecal samples. Further evaluation of processing and decontamination steps, culture conditions, and incubation period is needed for testing samples from sheep and other species of susceptible animals. In addition work on conventional versus real-time PCR and optimization of suitable DNA extraction protocols should be continued further to determine the extent of the differences in the results one might get if these are varied.

	Acknowledgments

 
The work was performed at Animal Disease Diagnostic Laboratory, Ohio Department of Agriculture Reynoldsburg OH 43068 using funding from USDA/APHIS Grant No. 03-9100-0804-GR. We thank Kristi Ott, Melissa Hart, and Troy Farrell for their excellent technical support. We would like to thank Dr. Charles Baldwin and Dr. Murray Hines, Veterinary Diagnostic and Investigational Laboratory for critically reviewing the manuscript.

	Sources and manufacturers

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From the University of Georgia, Veterinary Diagnostic and Investigational Laboratory, Tifton GA (Rajeev), and the Department of Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH (Shulaw), and the Department of Population Health and Reproduction, School of Veterinary Medicine, University of California - Davis, CA (Berghaus), and the Animal Disease Diagnostic Laboratory, Ohio Department of Agriculture, Reynoldsburg, OH (Zhang, Byrum).

^a. Trek Diagnostic Systems, Westlake OH.

^b. Becton Dickinson, Sparks, MD.

^c. IKA WORKS INC, Wilmington, NC.

^d. Pierce Chemicals, Rockford IL.

^e. Qiagen Sciences, MD.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rajeev, S. Articles by Byrum, B. Search for Related Content PubMed PubMed Citation Articles by Rajeev, S. Articles by Byrum, B.