Incidence of Mycobacterium paratuberculosis in Bulk Raw and Commercially Pasteurized Cows' Milk from Approved Dairy Processing Establishments in the United Kingdom

Scope of M. paratuberculosis milk testingDuring the 17-month period from March 1999 to July 2000 (inclusive), a total of 814 bulk raw and commercially pasteurized cows' milk samples from 241 approved dairy processing establishments throughout England, Wales, Scotland, and Northern Ireland were tested for the presence of M. paratuberculosis. Participation in the United Kingdom milk survey was on an entirely voluntary basis, 755 approved dairy establishments which heat treat milk (both drinking milk and milk for the manufacture of other dairy products) having been approached in advance of its commencement and 258 having agreed to take part. Care was taken by the FSA to ensure that the companies who agreed to take part were as representative as possible of the United Kingdom dairy processing sector in terms of dairy size, annual throughput, and geographic location. For M. paratuberculosis testing purposes, dairy processing establishments were visited by sampling officers on a single occasion, and a minimum of one raw milk sample and a number of heat-treated milk samples were collected. In total, 244 bulk raw and 567 pasteurized (228 whole, 179 semiskim, and 160 skim) milk samples were examined for the presence of M. paratuberculosis.

Sample collection and transport.A sample of raw milk was collected from a bulk raw milk silo at each dairy processing establishment, and a carton or cartons of pasteurized product were collected while coming off the production line. Sampling officers were instructed that whenever possible, the raw and pasteurized milk samples were to be taken from the same batch of milk.

Milk samples collected from approved dairy establishments in England and Wales were taken to ADAS Laboratories, Wolverhampton, United Kingdom, where the main microbiological analyses for milk samples from these regions were to be carried out. Each milk sample was assigned a numerical code, and a subsample was aseptically transferred into a sterile 150-ml plastic sample bottle for dispatch to QUB for M. paratuberculosis testing. Milk samples from dairy establishments throughout Scotland were taken to four testing laboratories, in Aberdeen, Glasgow, Edinburgh, and Dundee, where subsamples were taken for dispatch to QUB. All milk samples from the United Kingdom mainland were placed in insulated boxes containing ice packs to maintain the temperature of the samples at 4°C or below and were transported to the testing laboratory at QUB via overnight courier service. In Northern Ireland, Department of Agriculture and Rural Development sampling officers aseptically collected raw and pasteurized milk samples from local dairy processing establishments and delivered them directly to the testing laboratory at QUB.

M. paratuberculosis testing of all milk samples, irrespective of origin, commenced within 72 h of collection from the dairy processing establishment, and the temperature of the milk samples was maintained at or below 4°C at all times between collection and testing. No information regarding the origin of the milk samples accompanied the samples to QUB. Information on the origin of each milk sample, its corresponding sample code, and processing details for pasteurized milk samples (collected at the time of sampling) was held centrally by the FSA in London, United Kingdom, until the full results of M. paratuberculosis testing became available.

Milk testing.Each milk sample was subjected to two tests for M. paratuberculosis: IMS-PCR (to detect the presence of M. paratuberculosis cells, live or dead) and chemical decontamination and culture (to confirm the presence of viable M. paratuberculosis), as follows.

(i) IMS-PCR.A 50-ml portion of each raw milk sample was centrifuged (15 min at 2,500 × g), and the pellet was resuspended in 1 ml of phosphate-buffered saline (pH 7.4) containing 0.05% Tween 20 (PBS-T; Sigma Chemical Co. Ltd, Poole, Dorset, United Kingdom) prior to IMS, which was carried out as described previously (10). In an effort to maximize test sensitivity and produce consistently clear IMS-PCR results, samples were subjected to a modification of the previously described (12) DNA extraction and purification protocol after IMS and before PCR was carried out. This protocol involved overnight incubation of the resuspended beads after IMS at 37°C in 700 μl of TEN lysis buffer (2 mM EDTA, 400 mM NaCl, 10 mM Tris [pH 8.0], 0.6% sodium dodecyl sulfate) containing 20 μg of proteinase K (Sigma); mechanical disruption of the sample in blue-cap tubes in a Hybaid Ribolyser (both from Hybaid Ltd., Middlesex, United Kingdom); and extraction, purification, and precipitation of the DNA with phenol, chloroform-isoamyl alcohol (24:1), and isopropanol (all from Sigma), respectively. Precipitated DNA was washed once in 70% ethanol and resuspended in 50 μl of Tris-EDTA buffer before PCR was performed. Each PCR mixture consisted of 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 1.75 mM MgCl₂, 150 μM each deoxynucleoside triphosphate, 60 pmol each of primers P90 and P91 (23), and 1.25 U of Platinum Taq DNA polymerase (Life Technologies Ltd., Paisley, Scotland). The mixture was overlaid with 2 drops of light mineral oil. PCRs were carried out for 33 cycles of 94°C for 2 min, 58°C for 1 min, and 72°C for 1 min, with an initial denaturation step at 94°C for 10 min and a final annealing step at 72°C for 7 min. PCR products were visualized after 2% agarose gel electrophoresis by staining with ethidium bromide (0.5 μg/ml) and viewed on a UV transilluminator. The size of the amplified product was checked with molecular size markers (φX174 replicative-form DNA HaeIII fragments; Sigma). IMS-PCR results were reported as positive if a band of the correct size (400 bp) was observed on the gel. The minimum detection limit of this modified IMS-PCR protocol was estimated to be in the region of 1 CFU/50 ml of milk (unpublished data), a value which represents a substantial improvement in sensitivity over that of the original IMS-PCR protocol (10³ CFU/50 ml) (12); the latter protocol simply used boiling at 100°C for 15 min to release DNA between IMS and PCR.

(ii) Decontamination and culture.A second 50-ml portion of each milk sample was centrifuged (15 min at 2,500 × g), and the pellet was resuspended in 10 ml of freshly prepared 0.75% (wt/vol) cetylpyridinium chloride (HPC; Sigma). Following incubation at room temperature (21°C) for 5 h and further centrifugation (as described above), the pellet was resuspended in 1 ml of PBS-T. Two slopes of Herrold's egg yolk medium containing 2 μg of mycobactin J/ml (HEYM) were each inoculated with 250 μl of the resuspended pellet. One vial of BACTEC 12B radiometric medium (Becton Dickinson, Cowley, Oxford, England) supplemented with 0.5 ml of Difco egg yolk emulsion, 2 μg of mycobactin J (Synbiotics Europe SAS, Lyon, France)/ml, and PANTA antibiotic supplement (Becton Dickinson) reconstituted in accordance with the manufacturer's instructions (100 μl for raw milk cultures and 50 μl for pasteurized milk cultures) was inoculated with 500 μl of the resuspended pellet. Both media were incubated at 37°C for up to 18 weeks. Slopes were examined periodically for the presence of colonies. BACTEC vials were read regularly on a BACTEC 460TB instrument (Becton Dickinson). When growth was observed in either medium, acid-fast staining was carried out by the Ziehl-Neelsen method to confirm the presence of acid-fast organisms. Further confirmatory tests (14) to confirm slow growth rate, typical colony morphology, and mycobactin J dependence and IS 900 PCR of a sample from a colony were carried out on each suspect acid-fast isolate to determine whether it was M. paratuberculosis or some other Mycobacterium sp.

Statistical analysis of culture and IMS-PCR results.The number of confirmed M. paratuberculosis isolates or IMS-PCR-positive results obtained for each type of milk was expressed as a percentage of the total number of samples of that type of milk tested. The standard error of this value was calculated, and 95% confidence limits are presented.

Test controls.Negative (water only) and positive (DNA obtained from M. paratuberculosis strain B2) PCR controls and negative (PBS-T used to resuspend the milk pellet) and positive (M. paratuberculosis strain B2 broth culture) IMS controls were run alongside each batch of milk samples to confirm that both PCR and IMS were operating correctly. On three separate occasions during the survey, some raw cows' milk was obtained and spiked with M. paratuberculosis strain B2 (approximately 10⁴ CFU added per 100 ml of milk) to act as an internal method control. This spiked milk sample was subjected to both tests to confirm that M. paratuberculosis was isolated after decontamination and culture and detected after IMS-PCR. Spiked positive controls yielded typical colonies after HPC decontamination and culture and tested IMS-PCR positive on all three occasions.

Phosphatase testing.All pasteurized milk samples received for M. paratuberculosis testing at QUB were subjected to phosphatase testing upon receipt. Initially, the long-established Aschaffenberg-Mullen method was applied, but part way through the survey (December 1999 onward), this method was replaced by the Fluorophos method, which is capable of detecting much lower levels of residual phosphatase activity (20).

General laboratory control measures.Control measures were in place throughout the survey in order to prevent possible accidental laboratory contamination of the milk samples with M. paratuberculosis strains in use in the testing laboratory at QUB over the same period. (i) Milk samples were processed in a part of the category 3 pathogen laboratory physically removed from the class I safety cabinet to which all M. paratuberculosis research involving live M. paratuberculosis cultures was confined. (ii) Separate sets of dedicated pipette aids were used for IMS-PCR and culturing, and disposable filter tips were used at all times. (iii) IMS-dedicated pipette aids were regularly subjected to UV treatment to render any contaminating DNA nonamplifiable by PCR. (iv) HPC decontaminant solution and PBS-T solution (used during IMS and for resuspension of the milk pellet after decontamination) were freshly made on each day of testing, filter-sterilized (0.2-μm pore size), and divided into aliquots of the required volumes before use.

Molecular typing of confirmed M. paratuberculosis milk isolatesFourteen confirmed M. paratuberculosis milk isolates were typed at QUB by IS 900 restriction fragment length polymorphism (RFLP) typing with restriction enzyme BstEII broadly as described in the standardized protocol for the RFLP typing of M. paratuberculosis strains (24). DNA from four laboratory strains of M. paratuberculosis (ATCC 19698, B2, DVL 943, and NCTC 8578) that had been in use in the laboratory at QUB during the period of the survey was also RFLP typed. Briefly, DNA was obtained from the pellet of cells resulting from centrifugation of a 10-ml sample of a Middlebrook 7H9 broth culture of each strain by overnight proteinase K treatment in TEN lysis buffer, ribolysation, and extraction as described earlier for IMS-PCR. DNA quantitation standards (Life Technologies Ltd.) were used to quantify the yield of DNA and to standardize the amount of DNA subjected to treatment with restriction enzyme BstEII (Life Technologies Ltd.). DNA was digested at 37°C for 3 h before being applied to a 1% agarose gel along with lambda DNA HindIII molecular weight markers (Sigma). DNA fragments were separated by electrophoresis at 30 V for 16 h. After depurination, denaturation, and neutralization, the digested DNA in the gel was transferred to a Hybond N+ membrane (Amersham Pharmacia Biotech United Kingdom Ltd., Little Chalfont, Buckinghamshire, United Kingdom) by Southern blotting. The DNA was fixed to the membrane by UV cross-linking at 2 J/cm² with a Fluo-Link machine (Vilber Lourmat, Torcy, France). The membrane was hybridized overnight with an IS 900 probe generated in-house from DNA of type strain ATCC 19698 (as described by Pavlik et al. [24]) according to the instructions accompanying the ECL direct nucleic acid labeling kit (Amersham). RFLP profiles were visualized on Hyperfilm ECL (Amersham). RFLP profiles were visually compared to published profiles (24) and assigned to a particular RFLP type, whenever possible.

The 14 confirmed milk isolates and four laboratory strains were also independently typed by Karen Stevenson and Valerie Hughes at Moredun Research Institute near Edinburgh, Scotland, by using pulsed-field gel electrophoresis (PFGE) and three restriction enzymes, SnaBI, NdeI, and SpeI, in accordance with the method described recently by Hughes et al. (19).

Detection of M. paratuberculosis by IMS-PCRThe IMS-PCR results for raw and pasteurized milk samples tested during the survey are summarized in Table 1. Overall, M. paratuberculosis DNA was detected by IMS-PCR in 19 (7.8%; 95% confidence interval, 4.3 to 10.8%) of the raw and 67 (11.8%; 95% confidence interval, 9.0 to 14.2%) of the pasteurized (12.0% whole, 14.4% semiskim, and 8.1% skim) milk samples tested. The distribution of IMS-PCR-positive results by month of survey is shown in Fig. 1. In all except the final 2 months of the survey (June and July 2000), IMS-PCR-positive results were obtained. The percentage of IMS-PCR-positive samples ranged from 2 to 27.3% of samples tested per month (mean, 10.4%). The IMS-PCR findings suggest that 56 (23.5%) of the 241 United Kingdom dairy processing plants from which milk samples were submitted during the survey may have been processing milk contaminated by M. paratuberculosis. However, as not many of the IMS-PCR results were strongly positive, the organism is liable to have been present at low levels in the milk being processed.

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FIG. 1.

Distribution of IMS-PCR-positive results for raw and commercially pasteurized cows' milk by month of survey. The dashed line indicates the overall mean percent IMS-PCR-positive level.

Isolation of viable M. paratuberculosis by cultureThe culture results for raw and pasteurized milk samples tested during the survey are also summarized in Table 1. A total of 14 confirmed M. paratuberculosis isolates were obtained during the course of the milk survey, 4 from bulk raw milk samples and 10 from commercially pasteurized milk samples, representing 1.6% (95% confidence interval, 0.04 to 3.1%) of raw and 1.8% (95% confidence interval, 0.7 to 2.8%) of pasteurized milk samples, respectively. Overall, 2.8% of pasteurized semiskim, 1.3% of pasteurized whole, and 1.2% of pasteurized skim milk samples tested culture positive. On only one occasion did the raw and two different pasteurized milk samples from the same dairy processing establishment all test culture positive for M. paratuberculosis. Otherwise, there was no correlation between culture results for raw and pasteurized milk samples from the same dairy processing plant. Overall, pasteurized products from 8 (3.3%) of 241 United Kingdom dairy processing plants tested culture positive for M. paratuberculosis.

All 14 confirmed M. paratuberculosis isolates were obtained from HEYM slope cultures, and only 1 pasteurized milk sample was culture positive in BACTEC medium as well. Typically, only a small number of colonies (n = 1 to 5) were observed on HEYM cultures from M. paratuberculosis-positive milk samples. This finding translates to an M. paratuberculosis count in the original milk sample of about 4 to 20 CFU/50 ml of milk. As chemical decontamination was applied prior to culturing, this count is likely to be an underestimate of the number of M. paratuberculosis organisms actually present (7, 13). The one pasteurized milk sample that was culture positive with both HEYM and BACTEC medium yielded a lawn of typical colonies on HEYM slope cultures, suggesting the presence of larger numbers of M. paratuberculosis organisms in that particular sample. In addition to the 14 milk samples confirmed to contain viable M. paratuberculosis, two further pasteurized semiskim milk samples gave rise to BACTEC cultures that tested strongly acid fast and IS900 PCR positive after incubation. However, colonies were never obtained from these liquid cultures upon subculturing to HEYM, and so the presence of viable M. paratuberculosis could not be confirmed in these two samples.

Contamination or overgrowth of HEYM and BACTEC cultures may have accounted for the failure to isolate more culture-positive samples from raw milk in particular. Table 2 compares the culture medium contamination rates for raw and pasteurized milk cultures during the survey. HEYM contamination was considerably greater for raw milk cultures than for pasteurized milk cultures (27.8 and 6.9%, respectively). Conversely, fewer BACTEC cultures from raw milk (11.2%) than from pasteurized milk (26.4%) were contaminated, probably due to the addition of twice as much PANTA antibiotic supplement to raw milk cultures as to pasteurized milk cultures. However, we also suspect that a more general problem existed for much of the survey with the Difco egg yolk supplement inhibiting the growth of M. paratuberculosis in the BACTEC cultures, for some reason. This problem may explain the unexpectedly poor performance of the liquid BACTEC medium compared to the solid HEYM for the recovery of heat-injured M. paratuberculosis during this study.

Molecular typing of confirmed M. paratuberculosis milk isolatesRFLP profiles were obtained for the 14 milk isolates and four laboratory strains. Five different RFLP types were differentiated among the strains tested, and two distinct RFLP types were shown by two of the milk isolates (Fig. 2A). Twelve of the RFLP profiles obtained for the milk isolates were easily assigned to particular RFLP types described previously by Pavlik et al. (24); the remaining two profiles were not.

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FIG. 2.

Distributions of RFLP and PFGE types among the M. paratuberculosis milk isolates and laboratory strains. Strain designations, when they could be assigned, are indicated in parentheses.

PFGE profiles were obtained for the four laboratory strains and all except 1 of the 14 milk isolates. The 14th milk isolate did not yield sufficient DNA after culturing, despite several attempts, to permit PFGE analysis. Six different PFGE types were differentiated among the 13 milk isolates (Fig. 2B); three of these were distinct from the PFGE types exhibited by the four laboratory strains. Hence, the results of both RFLP typing and PFGE typing indicate that M. paratuberculosis types distinct from those of the laboratory strains in use in the milk testing laboratory at QUB existed among the milk isolates.

Processing details for culture-positive pasteurized milk samples.The culture-positive pasteurized milk samples were from dairy processing establishments in all four regions of the United Kingdom, and the majority of the pasteurized milk tested was intended for retail sale as liquid milk. Information regarding pasteurization time and temperature conditions and other treatments applied to the milk during processing, collected at the time of milk sampling from the eight dairy processing establishments which yielded culture-positive pasteurized milk samples, is summarized in Table 3. Seven of the 10 culture-positive milk samples had received a pasteurization treatment of 72 to 74°C for a minimum of 15 s; the remaining 3 had been treated at 72 to 75°C for a minimum of 25 s. Five of the pasteurized milk samples had been subjected to homogenization during production. All pasteurized milk samples received for M. paratuberculosis testing at QUB were confirmed to be phosphatase negative, indicating that proper pasteurization had been achieved during their production.