Experimental Validation of a Nested Polymerase Chain Reaction Targeting the Genetic Element ISMAP02 for Detection of Mycobacterium Avium Subspecies Paratuberculosis in Bovine Colostrum

Abstract

Colostrum samples experimentally inoculated with Mycobacterium avium subsp. paratuberculosis (MAP; strain K-10) at increasing concentrations between 1 × 10¹ and 1 × 10⁹ cells/ml were tested for recovery of MAP DNA using a nested ISMAP02 target polymerase chain reaction initially developed for detecting MAP DNA in fecal samples. The following detection rates were achieved for sample replicates inoculated with unsonicated MAP pure stock: 100% between 1 × 10⁷ and 1 × 10⁹ cells/ml, 75% between 1 × 10³ and 1 × 10⁶ cells/ml, and 50% between 1 × 10¹ and 1 × 10² cells/ml replicates. Detection rates achieved for the colostrum sample replicates inoculated with sonicated MAP cell suspension were 75% for 1 × 10⁹ cells/ml, 100% between 1 × 10⁷ and 1 × 10⁸ cells/ml, 75% for 1 × 10⁶ cells/ml, 0 for 1 × 10⁴ cells/ml, and 25% between 1 × 10¹ and 1 × 10³ cells/ml. When negative control colostrum samples were tested, 16 of 18 (89%) samples were correctly detected as negative for MAP DNA using the current assay. In conclusion, the MAP DNA detection rates of the present assay improved with increasing concentrations of MAP in the colostrum sample replicates, although MAP DNA was also detected in 2 of 18 (11%) negative control samples, suggesting an undefined technical problem with the assay or, perhaps, sample contamination during preparation. Overall, the present findings suggest a potential role of the proposed polymerase chain reaction assay to detect MAP in colostrum. However, adoption of this test for use in routine screening of field colostrum for MAP awaits findings from an ongoing field validation study.

Mycobacterium avium subsp. paratuberculosis (MAP) infection in cattle is characterized by chronic enteritis, progressive diarrhea, weight loss, and eventual death. Although the most common source of MAP is thought to be feces from infected adult cattle, with most transmissions occurring via the fecal–oral route, 13 MAP has also been detected in milk and colostrum collected from cows subclinically infected with Johne's disease, 12,14 suggesting that raw colostrum and waste milk might be some of the earliest sources of MAP by which calves become infected.

Although a number of factors are known to hinder efforts to control and ultimately eliminate Johne's disease from MAP-infected herds, including the long incubation period of the disease in cattle, the situation is compounded by limited availability of diagnostic assays with sufficient sensitivity to detect MAP-infected cattle early in the course of the disease. 3,9 The current commercially available diagnostic tests have been developed and occasionally field tested for direct and indirect detection of MAP in serum, milk, and fecal clinical specimens. 4,7,16,17 However, no validated diagnostic test for the direct or indirect detection of MAP in colostrum samples exists to date. The recent mapping of the complete MAP gene sequence 8 has provided an alternative involving the possible use of unique elements within the MAP genome for development of diagnostic assays. The genetic elements IS900 6 and ISMAP02 8 are considered specific to MAP and are detectable in clinical specimens by polymerase chain reaction (PCR). 3,11,15 In a 2005 study, 11 researchers developed and validated a nested PCR method targeting the multiple copy element ISMAP02 8 for detection of MAP in fecal samples.

In the present study, a previously developed nested ISMAP02 PCR 11 technique was validated for the detection of MAP in colostrum samples. The objectives of the current study were to estimate the limits of detection and to determine the ability of the nested ISMAP02 PCR to discriminate between known MAP-positive and -negative colostrum samples (negative controls). This technique could then be used in a future field-based study to determine the sensitivity and specificity of the assay.

Approximately 250 ml of fresh raw colostrum sample was collected from a single cow in a Minnesota dairy herd (45 adult cows) enrolled in the Minnesota Johne's Disease Control Program. The herds in the program are classified based on cattle types (i.e., beef or dairy) and program participation category (i.e., test-negative program or management program). Herds enrolled in the management program include those implementing a Johne's disease control effort, with or without herd testing for Johne's disease, and without classified herd status. Herds enrolled in the test-negative program include herds at levels 1, 2, 3, and 4 of the U.S. Voluntary Bovine Johne's Disease Control Program with each increase indicating a lower probability of Johne's disease presence in the herd. The herd selected in the present study had a level 4 herd status classification.

Prior to collection of the colostrum sample, blood and fecal samples were collected from the donor cow (adult cow ≥36 months old) and tested to determine her true MAP infection status as follows: 10 ml of blood was drawn from the coccygeal vein using a 20-gauge, 1-inch needle into a 16 mm × 100 mm blood collection tube a and tested for MAP antibodies using a previously described serologic method. 4 Additionally, 10 g of feces were collected from the rectum using disposable plastic rectal examination gloves and tested by a previously described conventional culture method for MAP. 17

The colostrum sample was collected as follows: 6 hr after calving, the teat skin of the donor cow was disinfected using 0.05% povidone iodine detergent and swabbed with 70% ethyl alcohol–impregnated pledgets prior to actual collection of colostrum samples. The fore stripping colostrum was discarded, and 250 ml of colostrum was collected into a sterile glass tube from all 4 quarters and stored at −20°C. The colostrum was transported to the National Animal Disease Center (U.S. Department of Agriculture, Agricultural Research Service, Ames, IA), for further processing and analysis. The ability of the nested ISMAP02 PCR to correctly discriminate between known MAP-positive and -negative colostrum samples was evaluated using a predetermined set of experimentally infected colostrum samples while unin-fected colostrum samples were used as controls.

The method for preparation of strain K-10 MAP pure cell suspensions used to experimentally inoculate the colostrum samples in the current study has been previously described. 11 Briefly, pure isolates of MAP were collected during the log growth phase (Abs_540nm = 0.2–0.4). Isolates were pelleted through centrifugation at 7,500 × g, washed twice with phosphate buffered saline (pH 7.4), and resuspended in phosphate buffered saline to a concentration of 1 × 10⁹ cells/ml. Half the preparation was sonicated b through short bursts at 35 W for between 10 and 15 sec to disperse potentially clustered bacterial particles. This was then followed by a 10-fold (1 × 10¹–1 × 10⁹ cells/ml) serial dilution of both sonicated and unsonicated stocks in phosphate buffered saline. One milliliter of colostrum samples in quadruplicate were then inoculated with the inocula preparations containing MAP in concentrations that ranged between 1 × 10¹ and 1 × 10⁹ cells/ml of sample.

Seventy-two colostrum samples were inoculated with strain K-10 cell suspension (i.e., 4 samples for each MAP [strain K-10] concentration level between 1 × 10¹ and 1 × 10⁹ cells/ml). The MAP (strain K-10) pure isolate used to inoculate 36 colostrum samples was sonicated to reduce potential clumping of the bacteria while the remaining 36 colostrum samples were spiked using the unsonicated MAP (strain K-10) pure isolate. Eighteen colostrum samples were not inoculated with MAP (strain K-10) and were used as negative controls.

For extraction of MAP DNA, 1 ml of colostrum was added to a sterile Eppendorf tube. Tubes were centrifuged at 15,000 × g for 5 min, and the whey was discarded. To each cell pellet, 1 mg of proteinase K solution c (10 mg/ml) was added, and samples were vigorously vortexed to resuspend the pellets. Samples were incubated overnight (15 hr) at 50°C in a shaking water bath. To each sample, 44 μl of TEN (10 mM Tris–HCl, pH 8.0; 1 mM ethylenediamine tetra-acetic acid, pH 8.0; 0.1 M NaCl) buffers d and 20 μl of 0.4 M NaOH were added, and samples were mixed by vortexing. Tubes were then placed in a boiling water bath for 30 min. After cooling, DNA was extracted by adding an equal volume of phenol, chloroform, and isoamyl alcohol e (25:24:1). The samples were vortexed followed by immediate centrifugation at 15,000 × g for 5 min. The aqueous layer was transferred to a new tube, and the DNA was precipitated by adding 2.2 volumes of cold 100% ethanol f and 0.1 volume of 3 M sodium acetate (pH 5.2). Samples were mixed gently by inversion and then placed in a −20°C freezer for 1 hr. The tubes were centrifuged at 15,000 × g for 15 min followed by decantation of the supernatants. The DNA pellets were air dried for 2–5 min, and then the pellets were resuspended in 20 μl of sterile water. The DNA was either used immediately or frozen at −20°C to be analyzed later.

Specific primers for the ISMAP02 element were selected for use in a nested PCR format for amplification of DNA isolated from the colostrum samples. The primer sequences for the initial amplification included 5'-GCACGGTTTTTCGGATAACGAG-3' (forward primer) and 5'-TCAACTGCGTCACGGTGTCCTG-3' (reverse primer). In the first round, PCR was run using a conventional thermal cycler. g The reaction mixture consisted of ultrapure distilled water (free of DNase and RNase), GeneAmp h 10 × PCR buffer II, 2.5 mM MgCl₂, 0.25 mM deoxynucleoside triphosphates, 0.3 μM primers, and 2 U DNA polymerase. i Negative controls consisted of the reaction mixture alone and the nonspiked colostrum sample collected from the cow previously confirmed to be MAP negative as mentioned earlier. The positive controls consisted of genomic DNA from MAP (strain K-10 isolates) and the colostrum samples spiked with the same MAP strain. Five microliters of DNA was added for each sample, and samples were run in triplicate in 96-well plates according to the following protocol: initial denaturing cycle at 94°C for 5 min, followed by 20 cycles at 94°C for 45 sec, 58°C for 1 min, and 72°C for 2 min, and a final extension cycle at 72°C for 7 min. 11 The second amplification was performed using real-time PCR j and included a fluorophore 6-carboxyfluorescein–labeled probe specific for the ISMAP02 target sequence for the semiquantitative evaluation of the test samples. Primers nested within the first set included 5'-GGATAACGAGACCGTGGATGC-3' (forward primer) and 5'-AACCGACGCCGCCAATACG-3' (reverse primer) for this second amplification, yielding a 117-bp product. 11 One microliter of DNA from the first amplification was added to a reaction mixture consisting of PCR mastermix, k ultrapure distilled water (free of DNase and RNase), 0.05 μM primers, and 0.05 μM of the fluorophore 6-carboxyfluorescein–labeled probe (5'-/56-fluorophore 6-carboxyfluorescein/CAACCCGCACGCTG/3BHQ-1/-3'). A standard was constructed by amplifying the ISMAP02 target from MAP strain K-10 genomic DNA and cloned into the topoisomerase I expression vector l followed by transformation in Escherichia coli. The insert was analyzed for accuracy after plasmid digestion with EcoRI and verification of size on a 4% agarose gel. Further verification of the cloned insert was conducted by sequencing the product using a commercial DNA analyzer m after labeling the product with BigDye Terminator v3.1 cycle sequencing kit. n Primers used for sequencing were M13F: 5'-CGTTGTAAAACGACGGCCAGT-3' (forward) and M13R: 5'-CAGGAAACAGCTATGAC-3' (reverse). Optimal concentrations of the plasmid for use as a real-time PCR standard ranged from 1 ng to 100 attg. Real-time PCR conditions for the amplification of test samples and standards were 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, 40 cycles at 95°C for 25 sec, and 60°C for 1 min. Positive samples were visualized in the form of sigmoid curves plotted on 2-dimensional grids, with the x-axis representing the PCR cycle number and the y-axis representing the relative fluorescence of the signal. Sample runs with threshold cycles in the range of 15–30 cycles were declared positive.

The detection rates for MAP were 100% between 1 × 10⁷ and 1 × 10⁹ cells/ml (4/4), 75% between 1 × 10³ and 1 × 10⁶ cells/ml (3/4), and 50% between 1 × 10¹ and 1 × 10² cells/ml (2/4) replicates for the colostrum samples inoculated with unsonicated strain K-10 cell suspensions. For the colostrum samples inoculated with sonicated strain K-10 cell suspensions, the detection rates were 75% for 1 × 10⁹ cells/ml (3/4), 100% between 1 × 10⁷ and 1 × 10⁸ cells/ml (4/4), 75% for 1 × 10⁶ cells/ml (3/4), 0% for 1 × 10⁴ cells/ml (0/4), and 25% between 1 × 10¹ and 1 × 10³ cells/ml (1/4) replicates. Lack of detection of DNA in colostrum samples inoculated with 1 × 10⁴ cells/ml of sonicated MAP cell suspension might have been due to an apparent absence of MAP organisms during serial dilution of the original inoculum due to the known clumping tendency of MAP and the fact that the sonication conditions may have been less than ideal to disperse clustered MAP cells.

The overall proportion of known MAP-positive colostrum samples (replicates) based on the unsonicated strain K-10 cell suspensions as the inocula correctly detected as positive for MAP DNA by the current assay was 28 of 36 (78%). However, when the colostrum samples inoculated with sonicated strain K-10 were evaluated, the proportion of colostrum samples (replicates) correctly detected as positive for MAP DNA was 19 of 36 (53%). The proportion of negative control samples correctly detected as negative for MAP DNA using the current protocol was 16 of 18 (89%).

Under laboratory conditions specified in the current study, the nested PCR protocol had detection limits for MAP that ranged between 1 × 10¹ and 1 × 10⁹ cells/ml in spiked colostrum samples, although the sensitivity was compromised at lower concentrations of the inoculum (i.e., only 25% and 50% of the colostrum samples containing between 1 × 10¹ and 1 × 10² cells/ml of sonicated and unsonicated strain K-10 cell suspensions were correctly identified as positive for MAP DNA using the present protocol).

Although 16 of 18 (89%) negative control samples were correctly identified as negative for MAP DNA using the present protocol, false-positive test outcomes were observed in 2 of 18 (11%) negative control samples. This finding, although not unexpected, was definitely disappointing. The nested aspect of the PCR assay was designed to improve specificity and sensitivity by amplifying target DNA in a 2-step procedure using 2 distinct primer pairs. However, it should be noted that the attempt at improving sensitivity comes at a great price in that the nested design detracts from specificity, increasing the likelihood of false-positive test outcomes as a result of the increased risk of crossover contamination. 1,2 In practice, running positive and negative controls through the entire process, including the DNA extraction process, is highly recommended to exclude potential contamination. 1 In the present study, negative controls consisting of the reaction mixture alone and positive controls consisting of genomic DNA from strain K-10 MAP isolates were run as controls to monitor the possibility of contamination and soundness of the current assay. Given that running a parallel test involving negative and positive controls in addition to the known MAP-positive colostrum samples did not reduce the likelihood of cross-contamination (i.e., false-positive outcomes), the results could have been different had the conventional PCR been employed instead. Therefore, further investigation is required regarding potential contamination of the assay.

Sonication through short bursts at 35 W for 10–15 sec to disperse potentially clustered mycobacterial cells appeared to compromise sensitivity of the current assay. This observation was contrary to initial expectation, because sonication has been previously shown to improve the efficiency of PCR detection of Gram-positive bacteria DNA under laboratory conditions by causing disruption of the cellular walls and subsequent release of DNA. 5 A previous study 10 demonstrated that sonication of MAP for 2 min at 100 W did not affect the viability of MAP and allowed for maximum colony-forming unit counts when cultured on Herrold's egg yolk medium. It is therefore likely that the sonication conditions used in the current study were insufficient to disperse clustered mycobacterial cells or improve efficiency of PCR detection, thus explaining the inconsistent and unexpected findings of the present study. Perhaps the results would have been different had the samples been sonicated at 100 W for 2 min. 10

Lastly, although the advantage of the present study lay in its laboratory-controlled nature, a major limitation was that the MAP (strain K-10) cell suspension concentrations (1 × 10¹–1 × 10⁹ cells/ml) used to spike the colostrum samples prior to analysis and the frequency of detection at the different concentration levels might not necessarily mimic the levels of MAP shed naturally in colostrum. Although data on the efficacy of this test based on field colostrum samples are currently lacking, work is being undertaken to determine the diagnostic sensitivity of this test on colostrum samples obtained from Minnesota dairy herds naturally infected with Johne's disease.

The present findings suggest a potential role of the proposed PCR assay to detect MAP in colostrum. However, adoption of this test for use in routine screening of field colostrum for MAP awaits findings from an ongoing field validation study.

Acknowledgements. The authors extend appreciation to Margaret Walker for technical assistance.