Evaluation of denaturing gradient gel electrophoresis for the detection of mycobacterial species and their potential association with waterborne pathogens.

The versatility of denaturing gradient gel electrophoresis (DGGE) protocol provides enough grounds for its wide application over an array of microorganisms. This work was designed to evaluate DGGE for the detection and confirmation of mycobacteria and their association, if any, with waterborne pathogens. A total of 76 samples comprising raw untreated water, schmutzdecke, floccules and final treated water obtained from a common water source, and two water treatment works (WTW1 and WTW2), were analysed. Thirty-five species were identified from the overall samples, with 7% (5/76), 13% (10/76) and 26% (20/76) from the common raw water source, WTW1 and WTW2 respectively. The majority of the species were Cyanobacteria, with high dominance in the raw water entering WTW2. In the final treated water of WTW1 Eutreptiella braarudii was found, and that of WTW2 contained Anabaena nereformis, Anabaena torulosa and Podocarpus nerrifolius. Furthermore, one Mycobacterium species was found in the raw water of WTW1 aside from the detection of Mycobacterium avium ssp. paratuberculosis by the technique. No association between mycobacteria and the other species was observed. This implies DGGE may be employed to study the diversity of other akin mycobacterial species from various sources, and not as a direct means of elucidating microbial associations.

Survival kinetics of Mycobacterium bovis during manufacture and ripening of raw milk Cheddar and Caerphilly cheese produced on a laboratory-scale.

AIMS: Persistence of Mycobacterium bovis was investigated in UK raw milk cheeses. METHODS AND RESULTS: Replicating traditional cheese production methods under stringent CL3 containment conditions, Cheddar and Caerphilly cheeses were produced with Myco. bovis inoculated raw milk. High-inoculum investigations used three Myco. bovis genotypes; later low-inoculum investigations used only Myco. bovis AF2122/97. High-inoculum Cheddar (n = 9) and Caerphilly (n = 9) were matured for a minimum of 12 and 4 months respectively; maturation of low-inoculum Cheddar (n = 3) and Caerphilly (n = 3) was up to 11 weeks. Survival of Myco. bovis was monitored by enumeration at different points throughout cheese manufacture and ripening. D values were calculated as follows: 57 and 59 days in high-inoculum Cheddar and Caerphilly, respectively, and 41 and 24 days in low-inoculum Cheddar and Caerphilly respectively. CONCLUSIONS: Mycobacterium bovis is concentrated in cheese curd and a proportion lost with the whey. Reduction in viability during manufacturing is limited, while significant Myco. bovis inactivation occurs during maturation. Inactivation was improved, during Caerphilly ripening, when acid development was enhanced by increasing the proportion of starter culture. SIGNIFICANCE AND IMPACT OF THE STUDY: Mycobacterium bovis inactivation data obtained could be used to inform assessment of the risk posed to consumers by raw milk dairy products.

Optimization of modified Middlebrook 7H11 agar for isolation of Mycobacterium bovis from raw milk cheese.

Reports have highlighted the absence of contemporary peer reviewed publications pertaining to Mycobacterium bovis culture from raw milk and cheese. By replicating traditional methods, cheese-making methodology and equipment were devised to produce Cheddar (n = 6) and Caerphilly (n = 3) artificially contaminated with M. bovis (three genotypes) under stringent laboratory-containment guidelines for handling hazardous microbiological material. Middlebrook 7H11, modified for M. bovis isolation, was assessed for capacity to enumerate M. bovis despite changing cheese microflora and prolonged M. bovis exposure to the cheese matrix using maturing cheese test portions (n = 63; up to 16 weeks). Malachite green (MG) containing media isolated M. bovis at significantly (P < 0·05) lower levels than unmodified Middlebrook 7H11 agar despite MG being a common adjunct of Middlebrook 7H11 agar modified for M. bovis growth. Subsequently, a selective MG-free Middlebrook 7H11 agar modified using haemolysed red cells and calf serum was demonstrated as the best performing (P < 0·05) medium for recovery of M. bovis from typical UK cheese types, Cheddar and Caerphilly. Significance and impact of the study: Following increased M. bovis infection of UK cattle, the risk posed to consumers from consumption of unpasteurized milk and dairy products has changed. Furthermore, published methods for the culture and molecular detection of M. bovis in raw milk products are limited. Cheese-making protocols and M. bovis culture media reported here provide tools for further investigation of M. bovis survival during all stages of cheese manufacture and could inform future assessment of the risk to consumers from M. bovis contamination of unpasteurized dairy products.

Detection of Mycobacterium avium ssp. paratuberculosis in cheese, milk powder and milk using IS900 and f57-based qPCR assays.

AIMS: To develop a quantitative PCR assay for sensitive and specific detection of Mycobacterium avium ssp. paratuberculosis (Map) in a range of dairy products. METHODS AND RESULTS: TaqMan(®) assays were designed to target the IS900 and f57 genetic elements of Map. Both real-time PCR assays were integrated with the Adiapure(®) Map DNA extraction kit and assessed separately for the detection/quantification of Map in spiked milk, Cheddar cheese and milk powder. Assays were validated against Cheddar cheese samples containing known concentrations of Map. The IS900 qPCR assay was significantly more sensitive than the assay based on the f57 primer/probe. At a threshold cycle value of 38, limits of detection (LOD) for the IS900 qPCR assay were 0·6 CFU ml(-1), 2·8 CFU g(-1) and 30 CFU g(-1) for artificially contaminated pasteurized milk, whole milk powder and Cheddar cheese, respectively. The respective LOD's for the f57 assay were 6·2 CFU ml(-1), 26·7 CFU g(-1) and 316 CFU g(-1). CONCLUSION: The integrated Adiapure(®) extraction - IS900 real time assay described is a sensitive, quantitative method for the detection of Map in dairy products. This is the first study to consider qPCR as a quantitative estimation of Map-DNA in cheese and whole milk powder. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay developed allows sensitive detection and quantification of Map DNA in a range of dairy products which is valuable for the screening and surveillance of this potential zoonotic organism.

Inactivation of Mycobacterium avium ssp. paratuberculosis in milk by UV treatment.

AIMS: To determine the effect of UV radiation on the viability of two strains of Mycobacterium avium ssp. paratuberculosis (Map) inoculated into milk. METHODS AND RESULTS: Mycobacterium avium ssp. paratuberculosis in a ultra heat treated milk matrix was subjected to increasing doses of UV-C radiation from 0 to 1836 mJ ml(-1) using a pilot-scale UV reactor (20 l capacity). Survival of Map was monitored by culture on Herrold's egg yolk medium, Middlebrook 7H10 medium and the FASTPlaqueTB phage assay. Differences in sensitivity to UV treatment were observed between strains, however, at 1000 mJ ml(-1) a Map kill rate of 0.1-0.6 log(10) was achieved regardless of strain used or method employed to enumerate Map. Although the inactivation trend was similar on the culture and phage assay, the former gave a consistently higher viable count. CONCLUSIONS: The use of UV radiation alone does not represent an alternative to current pasteurization regimes for a large reduction in viable Map in milk. SIGNIFICANCE AND IMPACT OF THE STUDY: To the authors' knowledge the work here represents the first pilot-scale UV treatment process used to assess UV efficacy to inactivate Map in milk. The results are similar to those obtained with a laboratory-scale process indicating the difficulties associated with UV treatment of an opaque liquid and the recalcitrance of Map towards inimical treatments.

Optimisation of decontamination method and influence of culture media on the recovery of Mycobacterium avium subspecies paratuberculosis from spiked water sediments.

The recovery of Mycobacterium avium subspecies paratuberculosis (Map) from the environment can be a laborious process - owing to Map being fastidious, its low number, and also high numbers of other microbial populations in such settings. Protocols i.e. filtration, decontamination and modified elution were devised to recover Map from spiked water sediments. Three culture media: Herrold's Egg Yolk Media (HEYM), Middlebrook 7H10 (M-7H10) and Bactec 12B were then employed to grow the organism following its elution. In the sterile sediment samples the recovery of Map was significant between the time of exposure for each of HEYM and M-7H10, and insignificant between both media (P < 0.05). However, in the non-sterile sediment samples, the HEYM grew other background microflora including moulds at all the times of exposure whilst 4 h followed by M-7H10 culture yielded Map colonies without any background microflora. Using sterile samples only for the Bactec 12B, the recovery of Map decreased as time of exposure increased. Based on these findings, M-7H10 should be considered for the recovery of Map from the natural environment including water sediments where the recovery of diverse microbial species remains a challenge. SIGNIFICANCE OF THE STUDY: Map is a robust pathogen that abides in the environment. In water treatment operations, Map associates with floccules and other particulate matter including sediments. It is also a fastidious organism, and its detection and recovery from the water environment is a laborious process and can be misleading within the abundance of other mycobacterial species owing to their close resemblance in phylogenetic traits. In the absence of a reliable recovery method, Map continues to pose public health risks through biofilm in household water tanks, hence the need for the development of a reliable recovery protocol to monitor the presence of Map in water systems in order to curtail its public health risks.

Effect of carbon dioxide on growth and extracellular enzyme production by Pseudomonas fluorescens B52.

The effect of carbon dioxide (30 mM.l-1) on growth and extracellular protease and lipase production by Pseudomonas fluorescens B52 growing in a simulated milk medium at 7 degrees C in fermenter was investigated. The addition of carbon dioxide was shown to have a differential effect with extracellular enzyme production, particularly lipase, being inhibited to a greater extent than bacterial growth and final cell numbers.

Incidence of Mycobacterium paratuberculosis in raw sheep and goats' milk in England, Wales and Northern Ireland.

A blind survey of 104 raw sheep and goats' milk samples (90 goat, 14 sheeps) from bulk tanks on farms throughout England, Wales and Northern Ireland was carried out over a 5-month period (January-May 1998) in order to determine the incidence of Mycobacterium paratuberculosis. Each milk sample (100 ml) was divided into two 50ml portions. One portion was decontaminated with 0.75% hexadecylpyridinium chloride for 5h before culture on slopes of Herrold's egg yolk medium and in BACTEC radiometric medium. The second portion was subjected to immunomagnetic separation followed by IS900 PCR (IMS-PCR). The IMS-PCR assay was employed in order to provide a more rapid indication of the presence of M. paratuberculosis in each milk sample than is possible by culture. Information on the Johne's disease status of the sheep and goat herds that took part in the survey was not sought at the time of milk sampling. However, it subsequently emerged that at least some of the herds whose bulk milk was tested during this study were previously or currently infected with Johne's disease. Overall, during this survey one raw goats' milk sample tested positive for the presence of M. paratuberculosis by IMS-PCR (<1% of milk samples tested) but no viable M. paratuberculosis were isolated by culture. The results of this study suggest that bulk raw sheep and goats' milk from these regions of the UK may not represent significant vehicles of transmission of M. paratuberculosis to humans.

Improved detection of Mycobacterium avium subsp. paratuberculosis In milk by immunomagnetic PCR.

The potential use of a novel immunomagnetic PCR (IMS-PCR) technique as a rapid method to screen milk samples for the presence of Mycobacterium avium subsp. paratuberculosis (M. ptb) was assessed. Immunomagnetic separation (IMS) for M. ptb, developed at Queen's University, Belfast, was applied to milk samples prior to IS900 PCR in order to selectively concentrate any M. ptb cells present and, at the same time, separate the cells from constituents of milk likely to inhibit subsequent PCR. This increased the sensitivity of IS900 PCR. IMS-PCR sensitivity could be further increased by initial centrifugation (2500 g for 20 min) of larger volumes of milk (10 and 50 ml), and resuspension of the sediment into a 1 ml volume appropriate for IMS treatment. Following IMS, template DNA for IS900 PCR was obtained by heating the bead-cell suspension in a thermal cycler at 100 degrees C for 15 min. It was estimated that the IMS-PCR assay could detect approximately 10(3)CFU of M. ptb per 50 ml of milk (equivalent to 20 CFU/ml), whereas the minimum detection limit of direct IS900 PCR was estimated at 10(5)CFU of M. ptb per 50 ml (equivalent to 2000 CFU/ml). A blind trial was carried out in which a total of 40 spiked (10(6)CFU M. ptb) and unspiked, raw and laboratory-pasteurised milk samples were independently tested by IMS-PCR and conventional IS900 PCR. IMS-PCR correctly identified 97. 5% of milk samples (sensitivity 100%, specificity 95%), including spiked milk samples before and after laboratory-pasteurisation. One false positive result was obtained which may have resulted from carryover between samples during the IMS procedure. Conventional IS900 PCR correctly identified only 72.5% of the same 40 milk samples (sensitivity 23%, specificity 100%). IMS-PCR was also shown to be capable of detecting natural M. ptb infection in raw sheep's milk, and raw and commercially pasteurised cows' milk.

Effect of nutrient starvation on the resistance of Escherichia coli O157:H7 to subsequent heat stress.

The resistance of three strains of Escherichia coli O157:H7 in their stationary growth phase to starvation (24 h in water at 37 degrees C) followed by a heat treatment (56 degrees C for up to 90 min) was determined. Starvation was found to increase significantly the resistance of two strains (NCTC 12079; eae+, VT1+, VT2+, and ATCC 43889 eae+, VT2+) but not the remaining strain (ATCC 43890 eae+, VT1+). Strain NCTC 12079 (only one tested) was shown to retain all of the three virulence factors after the two stresses. De novo protein synthesis was shown to be required for heat resistance. Evidence using an rpoS mutant indicated a central role for this gene in inducing heat resistance after a starvation stress. It is hoped that this work will contribute to more accurate risk assessments in certain food processing operations.

Iodixanol development of a laboratory-scale technique to monitor the persistence of Mycobacterium avium subsp. paratuberculosis in Cheddar cheese.

Mycobacterium avium subsp. paratuberculosis (Map) is a potential human pathogen known to be present in raw milk from infected dairy herds. Current pasteurisation regimes do not totally inactivate Map resulting in the possibility of viable cells being present in pasteurised milk used for Cheddar cheese production. A laboratory-based method, ensuring strict safety precautions, was developed to manufacture 800-g Cheddar blocks, experimentally contaminated (postpasteurisation) with two different strains of Map. The composition of the model Cheddar produced was consistent with commercial product. Syneresis of the cheese curd caused a 1 log10 concentration of Map numbers from milk to cheese for a strain isolated from pasteurised milk. The type strain NCTC 8578 did not show a similar concentration effect, but did however survive the Cheddar manufacturing process. A small percentage (<5%) of the Map load for each strain was recovered in the whey fraction during the process.

Occurrence of Mycobacterium avium subsp. paratuberculosis in raw water and water treatment operations for the production of potable water.

Mycobacterium avium subsp. paratuberculosis (Map) causes Johne's disease of cattle and is implicated as a cause of Crohn's disease in humans. The organism is excreted in animal faeces and can contaminate water catchment areas. This coupled with Map's survival in the environment means that water destined for domestic use may be a source of exposure. This work was designed to determine the occurrence of Map in Lough Neagh (the largest freshwater lake in the British Isles), used as a reservoir, and in two water treatment works (WTW1 and WTW2) which abstract from the lough and which have slow sand filtration (SSF) and dissolved air flotation respectively as their principal treatment regimes. The organism was not detected in lough water samples by culture (n=70) but 29% (20/70) were positive by PCR. In the raw water to WTW1 and WTW2 no culture positives were detected but 54% (13/24) and 58% (14/24) respectively were PCR positive. In WTW1 there were no culture positives at the SSF or final water but 31% (8/26) and 45% (9/20) respectively were PCR positive. In WTW2 similar results were obtained with 26% (6/23) and 48% (11/23) in the floccules and final water respectively. At WTW2 however one culture positive was detected in the final water. This latter finding is of concern. The inability to reach definitive conclusions indicates the need for further research, particularly in the detection methods for viable Map.

Occurrence and characteristics of cytotoxic necrotizing factors, cytolethal distending toxins and other virulence factors in Escherichia coli from human blood and faecal samples.

Escherichia coli isolates from human blood (n=266) and faecal (n=237) samples were examined for cytotoxic necrotizing factors 1 and 2 (CNF 1 and 2), cytolethal distending toxin (CDT), and putative virulence factors that have been associated with disease conditions in humans and animals. PCR showed that the chromosomally encoded, Rho-activating, CNF1 (68/544, 12.5%) was more common than the transmissible plasmid-borne CNF2 (3/544, 0.6%). The relative risk of having either CNF or CDT toxin genes in blood compared to faecal isolates was 3.88 (95% CI 2.36-6.38). This was highly significant (P<0.0001) and demonstrates the importance of these factors in bloodstream infections. Fifty-one of 65 (78%) E. coli bearing CNF1 and 11 of 21 (52%) of E. coli bearing CDT also carried the pyelonephritis-associated pilus gene, papG. The S fimbrial adhesin gene, sfa, was found in 57 blood (21%) and eight faecal samples (3%). The F17 fimbrial adhesin gene and afimbrial adhesin gene afa did not occur frequently. Haemolysin (hly) was found in all of the isolates tested. Further studies must be designed to identify the clinical significance of these genes and their role in pathogenesis.

An inter-laboratory ring trial for the detection and isolation of Mycobacterium avium subsp. paratuberculosis from raw milk artificially contaminated with naturally infected faeces.

There is a need for standardised, robust, reproducible molecular and culture methods to achieve clarification of the inactivation of Mycobacterium avium subsp. paratuberculosis (Map), the causative microbial agent of Johne's disease, in (faecally) contaminated milk and other food products such as meat. This study assessed the performance of a commercially available Map DNA extraction kit for milk Adiapure and accompanying PCR detection kit Adiavet alongside 'in-house' molecular and culture methods in an inter-laboratory ring trial using raw milk spiked with Map-infected faeces. The combined Adiapure-Adiavet Map DNA extraction and detection kit consistently detected 30 copies of IS900 (equivalent to approximately 2 cells) ml(-1) raw milk, when used in four different laboratories. Improvements in sensitivity and ease of use for 'in-house' Map detection were observed when the Adiapure extraction kit was combined with 'in-house' detection assays. Detection by real-time PCR methods, using the commercial extraction and detection systems, resulted in an overall detection rate of 100%, 90%, 85% and 25% for respective Map concentrations of 300, 30, 3 and 0.3 copies of IS900ml(-1) raw milk. Map, at 300 copies of IS900 (equivalent to approximately 20 Map cells) ml(-1) raw milk, was recovered from all samples cultured in mycobacteria growth indicator tube (MGIT) medium, from 10 of 12 samples on Herrold's egg yolk medium (HEYM) and not recovered from any samples using BACTEC medium. In conclusion, the Adiapure DNA extraction kit allows for sensitive and easy detection of Map in raw milk. The extraction method can form a candidate part of essential methodology and real-time PCR can further increase the sensitivity of the detection method. Moreover, MGIT medium is promising for culture-dependent detection of Map from raw milk.

Effect of high pressure and pasteurization on Mycobacterium avium ssp. paratuberculosis in milk.

AIMS: To determine the effect of high pressures alone and in conjunction with pasteurization on the viability of two strains of Mycobacterium avium ssp. paratuberculosis (Map). METHODS AND RESULTS: Map in a milk matrix was subjected to 400, 500 and 600 MPa with and without pasteurization (72 degrees C for 15 s) and plated onto Herrold's egg yolk medium (HEYM) and Middlebrook 7H10 (7H10) agar, both containing antibiotic supplements. Medium 7H10 was found to give a significantly (P < 0.001) better recovery than HEYM. A significantly greater (P < 0.001) reduction in viable numbers was observed using 500 MPa (mean log reduction of 6.52) compared with 400 MPa (mean log reduction of 2.56) and between 400 MPa and control (no applied pressure) for 10 min treatments. A treatment time of 10 min resulted in significantly (P < 0.001) fewer survivors than 5 min. Low numbers of survivors were still detected when pressure treatment at 400 and 600 MPa was combined with subsequent pasteurization. CONCLUSIONS: The use of high-pressure was effective in reducing viable numbers of Map but even when combined with pasteurization there were still survivors, albeit when high inoculum levels of Map were used. SIGNIFICANCE AND IMPACT OF THE STUDY: To the authors' knowledge the work reported here represents the first study of the efficacy of high-pressure treatments alone and in combination with pasteurization to kill Map. The results indicate that further research is warranted before more commercial-scale studies are commissioned.

Mycobacterium avium ssp. paratuberculosis and its potential survival tactics.

Mycobacterium avium ssp. paratuberculosis (Map) is an important animal pathogen with a potential, but as yet unproven, role in human disease. This review briefly describes the characteristics of Map that distinguish it from other Mycobacterium spp., presenting new information arising from completion of the sequencing of the Map genome. It then focuses on the potential mechanisms Map might employ to survive and disseminate in the environment, including interaction with protozoa and insects, dormancy, biofilm formation and aerosolization.

Characteristics of cytotoxic necrotizing factor and cytolethal distending toxin producing Escherichia coli strains isolated from meat samples in Northern Ireland.

Swabs collected from pig, lamb and beef carcasses and samples of pork, lamb and beef mince were cultured for Escherichia coli strains. Strains harbouring cytotoxic necrotizing factors (CNF1 and 2) and cytolethal distending toxins (CDT-I,-II,-III and -IV) were identified in plate cultures of the isolates by colony hybridization with labelled probes and multiplex PCR assays. Simplex and multiplex PCR assays were used to further characterize the isolates to determine the presence of P, S and F17 fimbriae as well as afimbrial adhesins and haemolysin. The serotype was also determined where possible. Thirty strains with the capacity to code for CNF (4), CDT (24) or both (2) were isolated and characterized, and a wide range of associated factor patterns was observed. The methods utilized were successful in demonstrating the detection of viable strains with potentially significant pathogenic factors from human food sources.

Development of an IMS-PCR assay for the detection of Mycobacterium avium ssp. paratuberculosis in water.

AIMS: To develop a sensitive detection method for Mycobacterium avium ssp. paratuberculosis (Map) in water by modifying and optimizing an existing immunomagnetic separation polymerase chain reaction (IMS-PCR) technique. METHODS AND RESULTS: Sterile distilled water (50 ml) spiked with 10(6) Map ml(-1) was subjected to either filtration (0.45 microm pore size) followed directly by IS900 PCR (method 1) or centrifugation (2500 g for 20 min) followed by IMS and IS900 PCR (method 2). Method 2 permitted the detection of Map, whereas method 1 did not. Method 2 was then optimized by adding different concentrations of Tween 80 (0.05, 0.1, 0.2, 0.4 and 0.6% v/v) to water samples spiked with Map (10(6)-1 CFU ml(-1)) prior to centrifugation, and assessing the impact of this action on the detection sensitivity of subsequent IMS-PCR. The optimum Tween 80 concentration was found to be 0.4%, which permitted the detection of 10 Map CFU ml(-1) in spiked water samples by IMS-PCR. CONCLUSIONS: This method will be used to determine the incidence of Map in water destined for domestic use in future studies. SIGNIFICANCE AND IMPACT OF THE STUDY: A sensitive method for the detection of Map in water involving addition of 0.4% Tween 80, centrifugation and IMS-PCR was developed.

A PCR assay for the detection of Campylobacter jejuni and Campylobacter coli in water.

A PCR assay has been developed for the detection of Campylobacter jejuni and Camp. coli in water samples. The sample is filtered through a membrane which is subjected to sonication to release the impacted cells. After removal of the filter from the cell suspension and a freeze/thaw cell lysis step, a semi-nested PCR is carried out on the filtrate using the primers CF02, CF03 and CF04 (Camp. jejuni flaA and flaB gene sequences). Incorporation of a sonication stage allows removal of the filter membrane since they have been shown to inhibit the PCR. In experiments with spike water samples (20 ml) a theoretical sensitivity of 10-20 campylobacter cells ml-1 was achieved. Using a sample volume of 100 ml this sensitivity can be increased to approximately 2 campylobacter cells ml-1.

Effect of chemical decontamination and refrigerated storage on the isolation of Mycobacterium avium subsp. paratuberculosis from heat-treated milk.

AIMS: To assess the impact of chemical decontamination and refrigerated storage before culture on the recovery of Mycobacterium avium subsp. paratuberculosis from heat-treated milk. METHODS AND RESULTS: Five-millilitre samples of ultra heat-treated (UHT) milk spiked with Myco. paratuberculosis NCTC 8578, B4 or 806R (ca 10(6) CFU ml(-1)) were heated at 63 degrees C for 20 or 30 min by submersion in a water bath. Heat-treated milk (0.5 ml) was cultured immediately into BACTEC 12B medium or refrigerated at 4 degrees C for 48 h before culture. Milk samples that received a 20-min heat treatment were also subjected to decontamination with 0.75% cetylpyridinium chloride (CPC) for 5 h at room temperature before inoculation into BACTEC 12B medium when tested immediately and after 48 h at 4 degrees C. BACTEC vials were monitored for evidence of growth over an 18-week incubation period at 37 degrees C. CPC decontamination resulted in a significant reduction in the number of culture-positive milk samples recovered immediately after heating (P < 0.05) and after refrigerated storage for 48 h (P < 0.01). Refrigerated storage for 48 h before testing did not have any significant effect, beneficial or detrimental, on Myco. paratuberculosis recovery rates. CONCLUSIONS: CPC decontamination applied to milk immediately or 48 h after heating will adversely affect the recovery of viable Myco. paratuberculosis, possibly leading to nonrecovery of the organism although viable cells are present in the original milk sample. SIGNIFICANCE AND IMPACT OF THE STUDY: Published pasteurization studies in which milk samples were decontaminated before culture will have underestimated the survival capability of Myco. paratuberculosis after high-temperature, short-time pasteurization. CPC decontamination should not be applied to pasteurized milk in future studies.